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  • 1.
    Alikhani, Nyosha
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berglund, Anna-Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Engmann, Tanja
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Voegtle, F. -Nora
    Pavlov, Pavel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Meisinger, Chris
    Langer, Thomas
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 410, nr 3, s. 400-410Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.

  • 2. Bano-Polo, Manuel
    et al.
    Martinez-Gill, Luis
    Wallner, Björn
    Nieva, Jose L.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Mingarro, Ismael
    Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 4, s. 830-840Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    alpha-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

  • 3.
    Berglund, Anna-Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pujol, Calire
    Duchene, Anne-Marie
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts2009Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 393, nr 4, s. 803-814Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated the targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein. The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import (i.e., into a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles). In vivo import was investigated as transient expression of the green fluorescent protein constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portions of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) are required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines) but very high number of hydroxylated residues (26–51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import, showing that in this case, the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS and LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs; in most cases, the N-terminal portion is essential for import into both organelles, but in a few cases, a domain structure was observed.

  • 4.
    Beskow, Anne
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Grimberg, Kristian Björk
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Bott, Laura C.
    Karolinska Institutet, Institutionen för cell- och molekulärbiologi.
    Salomons, Florian A.
    Karolinska Institutet, Institutionen för cell- och molekulärbiologi.
    Dantuma, Nico P.
    Karolinska Institutet, Institutionen för cell- och molekulärbiologi.
    Young, Patrick
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation2009Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 394, nr 4, s. 732-46Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97(Ufd1/Npl4) complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.

  • 5.
    Björklund, Åsa K.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ekman, Diana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Light, Sara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Frey-Skött, Johannes
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Domain Rearrangements in Protein Evolution2005Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 353, nr 4, s. 911-923Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most eukaryotic proteins are multi-domain proteins that are created from fusions of genes, deletions and internal repetitions. An investigation of such evolutionary events requires a method to find the domain architecture from which each protein originates. Therefore, we defined a novel measure, domain distance, which is calculated as the number of domains that differ between two domain architectures. Using this measure the evolutionary events that distinguish a protein from its closest ancestor have been studied and it was found that indels are more common than internal repetition and that the exchange of a domain is rare. Indels and repetitions are common at both the N and C-terminals while they are rare between domains. The evolution of the majority of multi-domain proteins can be explained by the stepwise insertions of single domains, with the exception of repeats that sometimes are duplicated several domains in tandem. We show that domain distances agree with sequence similarity and semantic similarity based on gene ontology annotations. In addition, we demonstrate the use of the domain distance measure to build evolutionary trees. Finally, the evolution of multi-domain proteins is exemplified by a closer study of the evolution of two protein families, non-receptor tyrosine kinases and RhoGEFs.

  • 6.
    Björklund, Åsa K.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Light, Sara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sagit, Rauan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nebulin: A Study of Protein Repeat Evolution2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, nr 1, s. 38-51Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein domain repeats are common in proteins that are central to the organization of a cell, in particular in eukaryotes. They are known to evolve through internal tandem duplications. However, the understanding of the underlying mechanisms is incomplete. To shed light on repeat expansion mechanisms, we have studied the evolution of the muscle protein Nebulin, a protein that contains a large number of actin-binding nebulin domains. Nebulin proteins have evolved from an invertebrate precursor containing two nebulin domains. Repeat regions have expanded through duplications of single domains, as well as duplications of a super repeat (SR) consisting of seven nebulins. We show that the SR has evolved independently into large regions in at least three instances: twice in the invertebrate Branchiostoma floridae and once in vertebrates. In-depth analysis reveals several recent tandem duplications in the Nebulin gene. The events involve both single-domain and multidomain SR units or several SR units. There are single events, but frequently the same unit is duplicated multiple times. For instance, an ancestor of human and chimpanzee underwent two tandem duplications. The duplication junction coincides with an Alu transposon, thus suggesting duplication through Alu-mediated homologous recombination. Duplications in the SR region consistently involve multiples of seven domains. However, the exact unit that is duplicated varies both between species and within species. Thus, multiple tandem duplications of the same motif did not create the large Nebulin protein. Finally, analysis of segmental duplications in the human genome reveals that duplications are more common in genes containing domain repeats than in those coding for nonrepeated proteins. In fact, segmental duplications are found three to six times more often in long repeated genes than expected by chance. 

  • 7.
    Covarrubias, Adrian Suarez
    et al.
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Högbom, Martin
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Bergfors, Terese
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Carroll, Paul
    Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London.
    Mannerstedt, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Oscarson, Stefan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Parish, Tanya
    Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London.
    Jones, T Alwyn
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Mowbray, Sherry L
    Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
    Structural, biochemical, and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis.2008Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 381, nr 3, s. 622-33Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C(gamma) in the formation of the alphabeta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth; such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.

  • 8.
    Cymer, Florian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    White, Stephen H.
    Mechanisms of Integral Membrane Protein Insertion and Folding2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 5, s. 999-1022Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The biogenesis, folding, and structure of alpha-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons-often referred to as protein-conducting channels-for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. (C) 2014 Elsevier Ltd. All rights reserved.

  • 9.
    Ekman, Diana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Björklund, Åsa K.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Quantification of the Elevated Rate of Domain Rearrangements in Metazoa2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, nr 5, s. 1337-1348Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most eukaryotic proteins consist of multiple domains created through gene fusions or internal duplications. The most frequent change of a domain architecture (DA) is insertion or deletion of a domain at the N or C terminus. Still, the mechanisms underlying the evolution of multidomain proteins are not very well studied.

    Here, we have studied the evolution of multidomain architectures (MDA), guided by evolutionary information in the form of a phylogenetic tree. Our results show that Pfam domain families and MDAs have been created with comparable rates (0.1–1 per million years (My)). The major changes in DA evolution have occurred in the process of multicellularization and within the metazoan lineage. In contrast, creation of domains seems to have been frequent already in the early evolution. Furthermore, most of the architectures have been created from older domains or architectures, whereas novel domains are mainly found in single-domain proteins. However, a particular group of exon-bordering domains may have contributed to the rapid evolution of novel multidomain proteins in metazoan organisms. Finally, MDAs have evolved predominantly through insertions of domains, whereas domain deletions are less common.

    In conclusion, the rate of creation of multidomain proteins has accelerated in the metazoan lineage, which may partly be explained by the frequent insertion of exon-bordering domains into new architectures. However, our results indicate that other factors have contributed as well.

  • 10.
    Ekman, Diana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Björklund, Åsa K.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Frey-Skött, Johannes
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Multi-domain Proteins in the Three Kingdoms of Life: Orphan Domains and Other Unassigned Regions2005Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 348, nr 1, s. 241-243Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Comparative studies of the proteomes from different organisms have provided valuable information about protein domain distribution in the kingdoms of life. Earlier studies have been limited by the fact that only about 50% of the proteomes could be matched to a domain. Here, we have extended these studies by including less well-defined domain definitions, Pfam-B and clustered domains, MAS, in addition to Pfam-A and SCOP domains. It was found that a significant fraction of these domain families are homologous to Pfam-A or SCOP domains. Further, we show that all regions that do not match a Pfam-A or SCOP domain contain a significantly higher fraction of disordered structure. These unstructured regions may be contained within orphan domains or function as linkers between structured domains. Using several different definitions we have re-estimated the number of multi-domain proteins in different organisms and found that several methods all predict that eukaryotes have approximately 65% multi-domain proteins, while the prokaryotes consist of approximately 40% multi-domain proteins. However, these numbers are strongly dependent on the exact choice of cut-off for domains in unassigned regions. In conclusion, all eukaryotes have similar fractions of multidomain proteins and disorder, whereas a high fraction of repeating domain is distinguished only in multicellular eukaryotes. This implies a role for repeats in cell-cell contacts while the other two features are important for intracellular functions.

  • 11.
    Ekman, Diana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Identifying and Quantifying Orphan Protein Sequences in Fungi2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, nr 2, s. 396-405Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    For large regions of many proteins, and even entire proteins, no homology to known domains or proteins can be detected. These sequences are often referred to as orphans. Surprisingly, it has been reported that the large number of orphans is sustained in spite of a rapid increase of available genomic sequences. However, it is believed that de novo creation of coding sequences is rare in comparison to mechanisms such as domain shuffling and gene duplication; hence, most sequences should have homologs in other genomes. To investigate this, the sequences of 19 complete fungi genomes were compared. By using the phylogenetic relationship between these genomes, we could identify potentially de novo created orphans in Saccharomyces cerevisiae. We found that only a small fraction, <2%, of the S. cerevisiae proteome is orphan, which confirms that de novo creation of coding sequences is indeed rare. Furthermore, we found it necessary to compare the most closely related species to distinguish between de novo created sequences and rapidly evolving sequences where homologs are present but cannot be detected. Next, the orphan proteins (OPs) and orphan domains (ODs) were characterized. First, it was observed that both OPs and ODs are short. In addition, at least some of the OPs have been shown to be functional in experimental assays, showing that they are not pseudogenes. Furthermore, in contrast to what has been reported before and what is seen for older orphans, S. cerevisiae specific ODs and proteins are not more disordered than other proteins. This might indicate that many of the older, and earlier classified, orphans indeed are fast-evolving sequences. Finally, >90% of the detected ODs are located at the protein termini, which suggests that these orphans could have been created by mutations that have affected the start or stop codons.

  • 12.
    Enquist, Karl
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fransson, Mawritz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Boekel, Carolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bengtsson, Inger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Geiger, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lang, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Aron
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johansson, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein2009Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 387, nr 5, s. 1153-1164Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.

  • 13.
    Groth, Petra
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Auslander, Simon
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Majumder, Muntasir Mamun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Schultz, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Johansson, Fredrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Petermann, Eva
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, nr 1, s. 70-82Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O-6-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-M-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gamma H2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.

  • 14.
    Hedin, Linnea E.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öjemalm, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bernsel, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hennerdal, Aron
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Illergård, Kristoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Enquist, Karl
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kauko, Anni
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cristobal, Susana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lerch-Bader, Mirjam
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, nr 1, s. 221-229Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

  • 15.
    Hegazy, Usama M.
    et al.
    National Research Centre (NRC), Egypt.
    Musdal, Yaman
    Hacettepe University, Turkey.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 9, s. 1509-1514Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kat and 300-fold increased K-M(GSH) values, displays an apparent Hill coefficient of 0.82 +/- 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency k(cat)/K-M(GSH) to near the wild-type value but still yields Hill coefficients of 0.61 +/- 0.08 and 0.86 +/- 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 +/- 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

  • 16.
    Heublein, Manfred
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ndi, Mama
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vazquez-Calvo, Carmela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vögtle, F.-Nora
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Alternative Translation Initiation at a UUG Codon Gives Rise to Two Functional Variants of the Mitochondria! Protein Kgd42019Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, nr 7, s. 1460-1467Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Kgd4 is a novel subunit of the mitochondria! a-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.

  • 17. Hubner, Isaac A.
    et al.
    Lindberg, Magnus
    Haglund, Ellinor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliverberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Shakhnovich, Eugene I.
    Common motifs and topological effects in the protein folding transition state.2006Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, ISSN 0022-2836, Vol. 359, nr 4, s. 1075-1085Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Through extensive experiment, simulation, and analysis of protein S6 (IRIS), we find that variations in nucleation and folding pathway between circular permutations are determined principally by the restraints of topology and specific nucleation, and affected by changes in chain entropy. Simulations also relate topological features to experimentally measured stabilities. Despite many sizable changes in Φ values and the structure of the transition state ensemble that result from permutation, we observe a common theme: the critical nucleus in each of the mutants share a subset of residues that can be mapped to the critical nucleus residues of the wild-type. Circular permutations create new N and C termini, which are the location of the largest disruption of the folding nucleus, leading to a decrease in both Φ values and the role in nucleation. Mutant nuclei are built around the wild-type nucleus but are biased towards different parts of the S6 structure depending on the topological and entropic changes induced by the location of the new N and C termini.

  • 18. Kaltofen, Sabine
    et al.
    Li, Chenge
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Huang, Po-Ssu
    Serpell, Louise C.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    André, Ingemar
    Computational De Novo Design of a Self-Assembling Peptide with Predefined Structure2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 2, s. 550-562Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein and peptide self-assembly is a powerful design principle for engineering of new biomolecules. More sophisticated biomaterials could be built if both the structure of the overall assembly and that of the self-assembling building block could be controlled. To approach this problem, we developed a computational design protocol to enable de novo design of self-assembling peptides with predefined structure. The protocol was used to design a peptide building block with a beta alpha beta fold that self-assembles into fibrillar structures. The peptide associates into a double beta-sheet structure with tightly packed a-helices decorating the exterior of the fibrils. Using circular dichroism, Fourier transform infrared spectroscopy, electron microscopy and X-ray fiber diffraction, we demonstrate that the peptide adopts the designed conformation. The results demonstrate that computational protein design can be used to engineer protein and peptide assemblies with predefined three-dimensional structures, which can serve as scaffolds for the development of functional biomaterials. Rationally designed proteins and peptides could also be used to investigate the subtle energetic and entropic tradeoffs in natural self-assembly processes and the relation between assembly structure and assembly mechanism. We demonstrate that the de novo designed peptide self-assembles with a mechanism that is more complicated than expected, in a process where small changes in solution conditions can lead to significant differences in assembly properties and conformation. These results highlight that formation of structured protein/peptide assemblies is often dependent on the formation of weak but highly precise intermolecular interactions.

  • 19.
    Kauko, Anni
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hedin, Linnea E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Thebaud, Estelle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cristobal, Susana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Repositioning of transmembrane alpha-helices during membrane protein folding2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, nr 1, s. 190-201Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt(Ph) glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.

  • 20.
    Kauko, Anni
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Illergård, Kristoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Coils in the membrane core are conserved and functionally important2008Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 380, nr 1, s. 170-180Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    With the increasing number of available α-helical transmembrane (TM) protein structures, the traditional picture of membrane proteins has been challenged. For example, reentrant regions, which enter and exit the membrane at the same side, and interface helices, which lie parallel with the membrane in the membrane–water interface, are common. Furthermore, TM helices are frequently kinked, and their length and tilt angle vary. Here, we systematically analyze 7% of all residues within the deep membrane core that are in coil state. These coils can be found in TM-helix kinks as major breaks in TM helices and as parts of reentrant regions.

    Coil residues are significantly more conserved than other residues. Due to the polar character of the coil backbone, they are either buried or located near aqueous channels. Coil residues are frequently found within channels and transporters, where they introduce the flexibility and polarity required for transport across the membrane. Therefore, we believe that coil residues in the membrane core, while constituting a structural anomaly, are essential for the function of proteins.

  • 21.
    Kemp, Grant
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kudva, Renuka
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    de la Rosa, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays2019Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, nr 6, s. 1308-1314Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have characterized the cotranslational folding of two small protein domains of different folds-the alpha-helical N-terminal domain of HemK and the beta-rich FLN5 filamin domain-by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (force-profile analysis, or FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, photo induced electron transfer, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.

  • 22.
    Klepsch, Mirjam
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kovermann, M.
    Low, C.
    Balbach, J.
    Permentier, H. P.
    Fusetti, F.
    de Gier, Jan-Willem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Slotboom, D. J.
    Berntsson, R. P. -A
    Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 414, nr 1, s. 75-85Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 angstrom) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 angstrom, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.

  • 23.
    Klepsch, Mirjam M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Persson, Jan O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen.
    de Gier, Jan-Willem L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 407, nr 4, s. 532-542Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.

  • 24. Koos, Björn
    et al.
    Kamali-Moghaddam, Masood
    David, Leonor
    Sobrinho-Simões, Manuel
    Dimberg, Anna
    Nilsson, Mats
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wählby, Carolina
    Söderberg, Ola
    Next-Generation Pathology-Surveillance of Tumor Microecology2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 11, s. 2013-2022Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    A tumor is a heterogeneous population of cells that provides an environment in which every cell resides in a microenvironmental niche. Microscopic evaluation of tissue sections, based on histology and immunohistochemistry, has been a cornerstone in pathology for decades. However, the dawn of novel technologies to investigate genetic aberrations is currently adopted in routine molecular pathology. We herein describe our view on how recent developments in molecular technologies, focusing on proximity ligation assay and padlock probes, can be applied to merge the two branches of pathology, allowing molecular profiling under histologic observation. We also discuss how the use of image analysis will be pivotal to obtain information at a cellular level and to interpret holistic images of tissue sections. By understanding the cellular communications in the microecology of tumors, we will be at a better position to predict disease progression and response to therapy.

  • 25.
    Lamb, John
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Jarmolinska, Aleksandra
    Michel, Mirco
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Menéndez-Hurtado, David
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Sulkowska, Joanna
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    PconsFam: An Interactive Database of Structure Predictions of Pfam Families2019Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, nr 13, s. 2442-2448Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    At present, about half of the protein domain families lack a structural representative. However, in the last decade, predicting contact maps and using these to model the tertiary structure for these protein families have become an alternative approach to gain structural insight. At present, reliable models for several hundreds of protein families have been created using this approach. To increase the use of this approach, we present PconsFam, which is an intuitive and interactive database for predicted contact maps and tertiary structure models of the entire Pfam database. By modeling all possible families, both with and without a representative structure, using the PconsFold2 pipeline, and running quality assessment estimator on the models, we predict an estimation for how confident the contact maps and structures are for each family.

  • 26. Law, Christopher J.
    et al.
    Almqvist, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för fysikalisk kemi, oorganisk kemi och strukturkemi.
    Bernstein, Adam
    Goetz, Regina M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för fysikalisk kemi, oorganisk kemi och strukturkemi.
    Huang, Yafei
    Soudant, Celine
    Laaksonen, Aatto
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för fysikalisk kemi, oorganisk kemi och strukturkemi.
    Hovmöller, Sven
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för fysikalisk kemi, oorganisk kemi och strukturkemi.
    Wang, Da-Neng
    Salt-bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT2008Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 378, nr 4, s. 828-839Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (Pi) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters.

  • 27.
    Lloris-Garcerá, Pilar
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Seppälä, Susanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Slusky, Joanna S. G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Rapp, Mikaela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Why Have Small Multidrug Resistance Proteins Not Evolved into Fused, Internally Duplicated Structures?2014Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, nr 11, s. 2246-2254Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The increasing number of solved membrane protein structures has led to the recognition of a common feature in a large fraction of the small-molecule transporters: inverted repeat structures, formed by two fused homologous membrane domains with opposite orientation in the membrane. An evolutionary pathway in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers has been posited. A gene duplication event enables the evolution of two oppositely orientated proteins that form antiparallel heterodimers. Finally, fusion of the two genes generates an internally duplicated transporter with two oppositely orientated membrane domains. Strikingly, however, in the small multidrug resistance (SMR) family of transporters, no fused, internally duplicated proteins have been found to date. Here, we have analyzed fused versions of the dual-topology transporter EmrE, a member of the SMR family, by blue-native PAGE and in vivo activity measurements. We find that fused constructs give rise to both intramolecular inverted repeat structures and competing intermolecular dimers of varying activity. The formation of several intramolecularly and intermolecularly paired species indicates that a gene fusion event may lower the overall amount of active protein, possibly explaining the apparent absence of fused SMR proteins in nature.

  • 28.
    Lloris-Garcerá, Pilar
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Slusky, Joanna S. G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Seppäla, Susanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Priess, Marten
    Schäfer, Lars V.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    In Vivo Trp Scanning of the Small Multidrug Resistance Protein EmrE Confirms 3D Structure Models2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 22, s. 4642-4651Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The quaternary structure of the homodimeric small multidrug resistance protein EmrE has been studied intensely over the past decade. Structural models derived from both two- and three-dimensional crystals show EmrE as an anti-parallel homodimer. However, the resolution of the structures is rather low and their relevance for the in vivo situation has been questioned. Here, we have challenged the available structural models by a comprehensive in vivo Trp scanning of all four transmembrane helices in EmrE. The results are in close agreement with the degree of lipid exposure of individual residues predicted from coarse-grained molecular dynamics simulations of the anti-parallel dimeric structure obtained by X-ray crystallography, strongly suggesting that the X-ray structure provides a good representation of the active in vivo form of EmrE.

  • 29.
    Lönneborg, Rosa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Smirnova, Irina
    Dian, Cyril
    Leonard, Gordon A
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    In vivo and in vitro investigation of transcriptional regulation by DntR.2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, nr 3, s. 571-82Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.

  • 30.
    Masson, Patrick
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Lundgren, Josefin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Young, Patrick
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Drosophila Proteasome Regulator REGγ: Transcriptional Activation by DNA Replication-related Factor DREF and Evidence for a Role in Cell Cycle Progression2003Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 327, nr 5, s. 1001-1012Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The proteasome regulator REG (PA28γ) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGγ in metazoan cells is currently unknown. To understand the role of Drosophila REGγ we have attempted to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGγ transcriptional assays were carried out with mutations in the proposed promoter. Our results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGγ transcription. To confirm that REGγ has a role in cell cycle progression, the effect of removing REGγ from S2 cells was tested using RNA interference. Drosophila cells depleted of REGγ showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGγ is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGγ was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila.

  • 31.
    Ndi, Mama
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Marin-Buera, Lorena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Salvatori, Roger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Singh, Abeer Prakash
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biogenesis of the bc(1) Complex of the Mitochondria! Respiratory Chain2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 21, s. 3892-3905Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The oxidative phosphorylation system contains four respiratory chain complexes that connect the transport of electrons to oxygen with the establishment of an electrochemical gradient over the inner membrane for ATP synthesis. Due to the dual genetic source of the respiratory chain subunits, its assembly requires a tight coordination between nuclear and mitochondrial gene expression machineries. In addition, dedicated assembly factors support the step-by-step addition of catalytic and accessory subunits as well as the acquisition of redox cofactors. Studies in yeast have revealed the basic principles underlying the assembly pathways. In this review, we summarize work on the biogenesis of the bc(1) complex or complex III, a central component of the mitochondrial energy conversion system.

  • 32.
    Nejedla, Michaela
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Li, Zhilun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Masser, Anna E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Biancospino, Matteo
    Spiess, Matthias
    Mackowiak, Sebastian D.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Friedländer, Marc R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Karlsson, Roger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    A Fluorophore Fusion Construct of Human Profilin I with Non-Compromised Poly(L-Proline) Binding Capacity Suitable for Imaging2017Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 429, nr 7, s. 964-976Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Profilin is vital for actin organisation in eukaryotic cells. It controls actin filament formation by binding monomeric actin and numerous proteins involved in polarised actin assembly. Important for the latter is the interaction surface formed by the N- and C-terminal helices, which pack close to each other on one side of the molecule at a distance from the actin site and mediate binding to poly-proline sequences present in many of the targeted proteins. Via these interactions, profilin contributes to the spatiotemporal control of actin filament growth. Studies of profilin dynamics in living cells by imaging techniques have been hampered by problems to generate fusion constructs with fluorophore proteins without negatively impacting on its poly-proline binding. With the object to circumvent this problem, we have generated an internal fusion of profilin with the green fluorescent variant citrine, here referred to as citrine profilin. The characterisation of citrine profilin (CIT-Pfn) demonstrates that it has full capacity to interact with poly-proline and also binds phosphatidylinositol lipids and actin, albeit with 10 times reduced affinity for the latter. Imaging of living cells expressing CIT-Pfn showed a distribution of the fusion protein similar to endogenous profilin. Furthermore, CIT-Pfn rescued the phenotypes observed after the Crispr/Cas9 knockout of the profilin 1 gene, including the lost migratory capacity characterising the knockout cells. Based on this, we conclude that the CIT-Pfn construct will be useful as a tool for displaying profilin localisation in living cells and obtaining information on its dynamic organisation under different conditions and activations of the actin microfilament and microtubule systems.

  • 33.
    Nilsson, IngMarie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lara, Patricia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hessa, Tara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Karamyshev, Andrey L.
    The Code for Directing Proteins for Trans location across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 6, s. 1191-1201Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the N-epsilon-(5-azido-2 nitrobenzoyl)-Lys-tRNA(amb) amber suppressor. A homogeneous population of SRP ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP signal sequence affinity and targeting to the translocon.

  • 34.
    Nilsson, Ola B.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Müller-Lucks, Annika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kramer, Günter
    Bukau, Bernd
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Trigger Factor Reduces the Force Exerted on the Nascent Chain by a Cotranslationally Folding Protein2016Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 428, nr 6, s. 1356-1364Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cotranslational protein folding can generate pulling forces on the nascent chain that can affect the instantaneous translation rate and thereby possibly feed back on the folding process. Such feedback would represent a new way of coupling translation and folding, different from coupling based on, for example, codon usage. However, to date, we have carried out the experiments used to measure pulling forces generated by cotranslational protein folding either in reconstituted in vitro translation systems lacking chaperones, in ill-defined cell lysates, or in vivo; hence, the effects of chaperones on force generation by folding are unknown. Here, we have studied the cotranslational folding of dihydrofolate reductase (DHFR) in the absence and in the presence of the chaperones trigger factor (TF) and GroEL/ES. DHFR was tethered to the ribosome via a C-terminal linker of varying length, ending with the SecM translational arrest peptide that serves as an intrinsic force sensor reporting on the force generated on the nascent chain when DHFR folds. We find that DHFR folds into its native structure only when it has emerged fully outside the ribosome and that TF and GroEL alone substantially reduces the force generated on the nascent chain by the folding of DHFR, while GroEL/ES has no effect. TF therefore weakens the possible coupling between cotranslational folding and translation.

  • 35. Norrgard, Malena A.
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-Site Residue Sustaining Catalytic Promiscuity2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 412, nr 1, s. 111-120Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The substrate-binding H-site of human glutathione transferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 mu mol min(-1) mg(-1) resulted primarily from an increased k(cat) value (70 s(-1)). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 mu mol min(-1) mg(-1). In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conserved Ile10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrate selectivity profiles in GSTs by a few mutations.

  • 36.
    Notari, Luigi
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Martínez-Carranza, Markel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Farías-Rico, José Arcadio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Cotranslational Folding of a Pentarepeat beta-Helix Protein2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 24, s. 5196-5206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a beta-helix pentarepeat protein from the human pathogen Clostridium botulinum a homolog of the fluoroquinolone resistance protein MfpA-using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight beta-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is similar to 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable beta-helix since it is structurally homologous to the smallest known beta-helix protein, a four-coil protein that is stable in solution.

  • 37.
    Nørholm, Morten H. H.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cunningham, Fiona
    Deber, Charles M.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Converting a Marginally Hydrophobic Soluble Protein into a Membrane Protein2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 407, nr 1, s. 171-179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    delta-Helices are marginally hydrophobic a-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between delta-helices and TM helices, we have studied the insertion of five TM-like delta-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated delta-helix is engineered into a bona fide membrane protein, we find that, for two delta-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a delta-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated delta-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect delta-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that delta-helices in secreted proteins are mutationally more distant from TM helices than delta-helices in cytosolic proteins.

  • 38. Orrell, Kathleen E.
    et al.
    Tellgren-Roth, Åsa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Di Bernardo, Mercedes
    Zhang, Zhifen
    Cuviello, Flavia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundqvist, Jasmin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Melnyk, Roman A.
    Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 18, s. 3190-3199Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Large clostridial toxins (LCTs) are a family of homologous proteins toxins that are directly responsible for the symptoms associated with a number of clostridial infections that cause disease in humans and in other animals. LCTs damage tissues by delivering a glucosyltransferase domain, which inactivates small GTPases, across the endosomal membrane and into the cytosol of target cells. Elucidating the mechanism of translocation for LCTs has been hampered by difficulties associated with identifying marginally hydrophobic segments that insert into the bounding membrane to form the translocation pore. Here, we directly measured the membrane-insertion partitioning propensity for segments spanning the putative pore-forming region using a translocon-mediated insertion assay and synthetic peptides. We identified membrane-inserting segments, as well as a conserved and functionally important negatively charged residue that requires protonation for efficient membrane insertion. We provide a model of the LCT pore, which provides insights into translocation for this enigmatic family of a-helical translocases.

  • 39. Ozenne, Valery
    et al.
    Noel, Jeffrey K.
    Heidarsson, Petur O.
    Brander, Soren
    Poulsen, Flemming M.
    Jensen, Malene Ringkjobing
    Kragelund, Birthe B.
    Blackledge, Martin
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Copenhagen.
    Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP2014Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, nr 3, s. 722-734Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.

  • 40.
    Peralvarez-Marin, Alex
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lorenz-Fonfria, Victor A.
    Bourdelande, Jose-Luis
    Querol, Enric
    Kandori, Hideki
    Padros, Esteve
    Inter-helical hydrogen bonds are essential elements for intra-protein signal transduction: The role of Asp115 in bacteriorhodopsin transport function2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 368, nr 3, s. 666-676Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The behavior of the D115A mutant was analyzed by time-resolved UV-Vis and Fourier transformed infrared (FTIR) spectroscopies, aiming to clarify the role of Asp115 in the intra-protein signal transcluctions occurring during the bacteriorhodopsin photocycle. UV-Vis data on the D115A mutant show severely desynchronized photocycle kinetics. FTIR data show a poor transmission of the retinal isomerization to the chromoprotein, evidenced by strongly attenuated helical changes (amide 1), the remarkable absence of environment alterations and protonation/deprotonation events related to Asp96 and direct Schiff base (SB) protonation form the bulk. This argues for the interactions of Asp115 with Leu87 (via water molecule) and Thr90 as key elements for the effective and vectorial proton path between Asp96 and the SB in the cytoplasmic half of bacteriorhodopsin. The results strongly suggest the presence of a regulation motif enclosed in helices C and D (Thr90-Pro91/Asp115) which drives properly the dynamics of helix C through a set of interactions. It also supports the idea that intra-helical hydrogen bonding clusters in the buried regions of transmembrane proteins can be potential elements in intra-protein signal transduction.

  • 41. Peschke, Markus
    et al.
    Le Goff, Mélanie
    Koningstein, Gregory M.
    Karyolaimos, Alexandros
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    de Gier, Jan-Willem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    van Ulsen, Peter
    Luirink, Joen
    SRP, FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjIC and Flk2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 3, s. 389-403Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs. In this study, we identify DjIC and Flk as bona fide Escherichia co/iTAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjIC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.

  • 42. Procko, Erik
    et al.
    Hedman, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hamilton, Keith
    Seetharaman, Jayaraman
    Fleishman, Sarel J.
    Su, Min
    Aramini, James
    Kornhaber, Gregory
    Hunt, John F.
    Tong, Liang
    Montelione, Gaetano T.
    Baker, David
    Computational Design of a Protein-Based Enzyme Inhibitor2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 18, s. 3563-3575Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.

  • 43.
    Schlegel, Susan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Löfblom, John
    Lee, Chiara
    Hjelm, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Klepsch, Mirjam
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Strous, Marc
    Drew, David
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)2012Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 423, nr 4, s. 648-659Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The 17 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the 17 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the 17 RNAP by the 17 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

  • 44.
    Schreiber, Fabian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Swedish E Sci Res Ctr, Stockholm, Sweden.
    Hieranoid: Hierarchical Orthology Inference2013Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 11, s. 2072-2081Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An accurate inference of orthologs is essential in many research fields such as comparative genomics, molecular evolution, and genome annotation. Existing methods for genome-scale orthology inference are mostly based on all-versus-all similarity searches that scale quadratically with the number of species. This limits their application to the increasing number of available large-scale datasets. Here, we present Hieranoid, a new orthology inference method using a hierarchical approach. Hieranoid performs pairwise orthology analysis using In Paranoid at each node in a guide tree as it progresses from its leaves to the root. This concept reduces the total runtime complexity from a quadratic to a linear function of the number of species. The tree hierarchy provides a natural structure in multi-species ortholog groups, and the aggregation of multiple sequences allows for multiple alignment similarity searching techniques, which can yield more accurate ortholog groups. Using the recently published orthobench benchmark, Hieranoid showed the overall best performance. Our progressive approach presents a new way to infer orthologs that combines efficient graph-based methodology with aspects of compute-intensive tree-based methods. The linear scaling with the number of species is a major advantage for large-scale applications and makes Hieranoid well suited to cope with vast amounts of sequenced genomes in the future. Hieranoid is an open source and can be downloaded at Hieranoid.sbc.su.se.

  • 45.
    Spiess, Matthias
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Echbarthi, Meriem
    Svanström, Andreas
    Karlsson, Roger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Grantham, Julie
    Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 17, s. 2757-2764Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta. (C) 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-ncV4.0/).

  • 46.
    Stenberg, Filippa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Assembly of the cytochrome bo3 complex2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 371, nr 3, s. 765-773Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An understanding of the mechanisms that govern the assembly of macromolecular protein complexes is fundamental for studying their function and regulation. With this in mind, we have determined the assembly pathway for the membrane-embedded cytochrome bo3 of Escherichia coli. We show that there is a preferred order of assembly, where subunits III and IV assemble first, followed by subunit I and finally subunit II. We also show that cofactor insertion catalyses assembly. These findings provide novel insights into the biogenesis of this model membrane protein complex.

  • 47.
    Stenmark, Pål
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dong, Min
    Dupuy, Jerome
    Chapman, Edwin R.
    Stevens, Raymond C.
    Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, nr 5, s. 1287-1297Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-angstrom X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  • 48.
    Stenmark, Pål
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Moche, Martin
    Gurmu, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordlund, Pär
    The Crystal Structure of the Bifunctional Deaminase/Reductase RibD of the Riboflavin Biosynthetic Pathway in Escherichia coli: Implications for the Reductive Mechanism2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, nr 1, s. 48-64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1–145) is similar to cytosine deaminase and the C-terminal domain (146–367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP+) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159–173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

  • 49.
    Ståhl, Annelie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, Stefan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Bhushan, Shashi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biverståhl, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Moberg, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Morisett, Magali
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vener, Alexander
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different2005Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, nr 4, s. 847-860Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P1P′1

    position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10–16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10–23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.

  • 50.
    Tarry, Michael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jaaskelainen, Mari
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Paino, Annamari
    Tuominen, Heidi
    Ihalin, Riikka
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Extra-Membranous Domains of the Competence Protein HofQ Show DNA Binding, Flexibility and a Shared Fold with Type I KH Domains2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, nr 4, s. 642-653Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Secretins form large oligomeric assemblies in the membrane that control both macromolecular secretion and uptake. Several Pasteurellaceae are naturally competent for transformation, but the mechanism for DNA assimilation is largely unknown. In Haemophilus influenzae, the secretin ComE has been demonstrated to be essential for DNA uptake. In closely related Aggregatibacter actinomycetemcomitans, an opportunistic pathogen in periodontitis, the ComE homolog HofQ is believed to be the outer membrane DNA translocase. Here, we report the structure of the extramembranous domains of HofQ at 2.3 angstrom resolution by X-ray crystallography. We also show that the extra-membranous domains of HofQ are capable of DNA binding. The structure reveals two secretin-like folds, the first of which is formed by means of a domain swap. The second domain displays extensive structural similarity to K homology (KH) domains, including the presence of a GxxG motif, which is essential for the nucleotide-binding function of KH domains, suggesting a possible mechanism for DNA binding by HofQ. The data indicate a direct involvement in DNA acquisition and provide insight into the molecular basis for natural competence.

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