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Patrick, J., Pettersson, P. & Mäler, L. (2023). Lipid- and substrate-induced conformational and dynamic changes in a glycosyltransferase involved in E. coli LPS synthesis revealed by 19F and 31P NMR. Biochimica et Biophysica Acta - Biomembranes, 1865(8), Article ID 184209.
Open this publication in new window or tab >>Lipid- and substrate-induced conformational and dynamic changes in a glycosyltransferase involved in E. coli LPS synthesis revealed by 19F and 31P NMR
2023 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1865, no 8, article id 184209Article in journal (Refereed) Published
Abstract [en]

WaaG is a glycosyltransferase (GT) involved in the synthesis of the bacterial cell wall, and in Escherichia coli it catalyzes the transfer of a glucose moiety from the donor substrate UDP-glucose onto the nascent lipopolysaccharide (LPS) molecule which when completed constitutes the major component of the bacterium's outermost defenses. Similar to other GTs of the GT-B fold, having two Rossman-like domains connected by a short linker, WaaG is believed to undergo complex inter-domain motions as part of its function to accommodate the nascent LPS and UDP-glucose in the catalytic site located in the cleft between the two domains. As the nascent LPS is bulky and membrane-bound, WaaG is a peripheral membrane protein, adding to the complexity of studying the enzyme in a biologically relevant environment. Using specific 5-fluoro-Trp labelling of native and inserted tryptophans and 19F NMR we herein studied the dynamic interactions of WaaG with lipids using bicelles, and with the donor substrate. Line-shape changes when bicelles are added to WaaG show that the dynamic behavior is altered when binding to the model membrane, while a chemical shift change indicates an altered environment around a tryptophan located in the C-terminal domain of WaaG upon interaction with UDP-glucose or UDP. A lipid-bound paramagnetic probe was used to confirm that the membrane interaction is mediated by a loop region located in the N-terminal domain. Furthermore, the hydrolysis of the donor substrate by WaaG was quantified by 31P NMR.

Keywords
glycosyltransferase, membrane interaction, substrate interaction, solution NMR, bicelle, lipids
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-220488 (URN)10.1016/j.bbamem.2023.184209 (DOI)001070814300001 ()37558175 (PubMedID)2-s2.0-85169551058 (Scopus ID)
Funder
Swedish Research Council, 621-2018-03395
Available from: 2023-08-29 Created: 2023-08-29 Last updated: 2023-10-10Bibliographically approved
Patrick, J., García Alija, M., Liebau, J., Pettersson, P., Metola, A. & Mäler, L. (2022). Dilute Bicelles for Glycosyltransferase Studies, Novel Bicelles with Phosphatidylinositol. Journal of Physical Chemistry B, 126(30), 5655-5666
Open this publication in new window or tab >>Dilute Bicelles for Glycosyltransferase Studies, Novel Bicelles with Phosphatidylinositol
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2022 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 126, no 30, p. 5655-5666Article in journal (Refereed) Published
Abstract [en]

Solution-state NMR can be used to study protein–lipid interactions, in particular, the effect that proteins have on lipids. One drawback is that only small assemblies can be studied, and therefore, fast-tumbling bicelles are commonly used. Bicelles contain a lipid bilayer that is solubilized by detergents. A complication is that they are only stable at high concentrations, exceeding the CMC of the detergent. This issue has previously been addressed by introducing a detergent (Cyclosfos-6) with a substantially lower CMC. Here, we developed a set of bicelles using this detergent for studies of membrane-associated mycobacterial proteins, for example, PimA, a key enzyme for bacterial growth. To mimic the lipid composition of mycobacterial membranes, PI, PG, and PC lipids were used. Diffusion NMR was used to characterize the bicelles, and spin relaxation was used to measure the dynamic properties of the lipids. The results suggest that bicelles are formed, although they are smaller than “conventional” bicelles. Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing PI and PC. The paramagnetic label was shown to have a shallow location in the bicelle, affecting the glycerol backbone of the lipids. We foresee that these bicelles will be useful for detailed studies of protein–lipid interactions. 

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-207911 (URN)10.1021/acs.jpcb.2c02327 (DOI)000838118300001 ()35880265 (PubMedID)2-s2.0-85135596847 (Scopus ID)
Available from: 2022-08-23 Created: 2022-08-23 Last updated: 2023-08-30Bibliographically approved
Brown, C., Patrick, J., Liebau, J. & Mäler, L. (2022). The MIT domain of chitin synthase 1 from the oomycete Saprolegnia monoica interacts specifically with phosphatidic acid. Biochemistry and Biophysics Reports, 30, Article ID 101229.
Open this publication in new window or tab >>The MIT domain of chitin synthase 1 from the oomycete Saprolegnia monoica interacts specifically with phosphatidic acid
2022 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 30, article id 101229Article in journal (Refereed) Published
Abstract [en]

Chitin synthases are vital for growth in certain oomycetes as chitin is an essential component in the cell wall of these species. In Saprolegnia monoica, two chitin synthases have been found, and both contain a Microtubule Interacting and Trafficking (MIT) domain. The MIT domain has been implicated in lipid interaction, which in turn may be of significance for targeting of chitin synthases to the plasma membrane. In this work we have investigated the lipid interacting properties of the MIT domain from chitin synthase 1 in Saprolegnia monoica. We show by fluorescence spectroscopy techniques that the MIT domain interacts preferentially with phosphatidic acid (PA), while it does not interact with phosphatidylglycerol (PG) or phosphatidylcholine (PC). These results strongly suggest that the specific properties of PA are required for membrane interaction of the MIT domain. PA is negatively charged, binds basic side chains with high affinity and its small headgroup gives rise to membrane packing defects that enable intercalation of hydrophobic amino acids. We propose a mode of lipid interaction that involves a combination of basic amino acid residues and Trp residues that anchor the MIT domain specifically to bilayers that contain PA.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-206192 (URN)10.1016/j.bbrep.2022.101229 (DOI)000832992800004 ()2-s2.0-85124297928 (Scopus ID)
Available from: 2022-06-22 Created: 2022-06-22 Last updated: 2022-08-24Bibliographically approved
Zhou, S., Pettersson, P., Björck, M. L., Dawitz, H., Brzezinski, P., Mäler, L. & Ädelroth, P. (2021). NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP. BMC Biology, 19(1), Article ID 98.
Open this publication in new window or tab >>NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP
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2021 (English)In: BMC Biology, E-ISSN 1741-7007, Vol. 19, no 1, article id 98Article in journal (Refereed) Published
Abstract [en]

Background: Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker's yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex.

Results: Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility.

Conclusions: Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

Keywords
ATP, Membrane protein, NMR, Solution structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-194982 (URN)10.1186/s12915-021-01036-x (DOI)000655036700002 ()33971868 (PubMedID)
Available from: 2021-07-28 Created: 2021-07-28 Last updated: 2024-01-17Bibliographically approved
Zhou, S., Pettersson, P., Huang, J., Brzezinski, P., Pomès, R., Mäler, L. & Ädelroth, P. (2021). NMR Structure and Dynamics Studies of Yeast Respiratory Supercomplex Factor 2. Structure, 29(3), 275-283
Open this publication in new window or tab >>NMR Structure and Dynamics Studies of Yeast Respiratory Supercomplex Factor 2
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2021 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 29, no 3, p. 275-283Article in journal (Refereed) Published
Abstract [en]

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc(1) and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble alpha helices and five putative transmembrane (TM) alpha helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation.

Keywords
charge zipper, protein-lipid interactions, molecular dynamics, mitochondria, Hig protein, membrane protein, solution structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-193384 (URN)10.1016/j.str.2020.08.008 (DOI)000629154700009 ()32905793 (PubMedID)
Available from: 2021-05-25 Created: 2021-05-25 Last updated: 2022-02-25Bibliographically approved
Pettersson, P., Patrick, J., Jakob, M., Jacobs, M., Klösgen, R. B., Wennmalm, S. & Mäler, L. (2021). Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter. Biochimica et Biophysica Acta - Biomembranes, 1863(2), Article ID 183529.
Open this publication in new window or tab >>Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter
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2021 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1863, no 2, article id 183529Article in journal (Refereed) Published
Abstract [en]

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer.

Keywords
Twin-arginine translocase, Oligomer, Vesicles, Membrane insertion, Membrane leakage, Electron microscopy, Light scattering, Chemical crosslinking, Circular dichroism, Fluorescence Correlation Spectroscopy
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-190036 (URN)10.1016/j.bbamem.2020.183529 (DOI)000603419900021 ()33279512 (PubMedID)
Available from: 2021-02-24 Created: 2021-02-24 Last updated: 2022-02-25Bibliographically approved
Rodrigo-Unzueta, A., Ghirardello, M., Urresti, S., Delso, I., Giganti, D., Anso, I., . . . Guerin, M. E. (2020). Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria. Biochemistry, 59(32), 2934-2945
Open this publication in new window or tab >>Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria
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2020 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 59, no 32, p. 2934-2945Article in journal (Refereed) Published
Abstract [en]

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-β-strand and β-strand-to-α-helix transitions and (ii) an “open-to-closed” motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the “open-to-closed” motion, with the β-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the “open-to-closed” motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-187850 (URN)10.1021/acs.biochem.0c00376 (DOI)000574868600004 ()32786405 (PubMedID)
Available from: 2020-12-16 Created: 2020-12-16 Last updated: 2022-02-25Bibliographically approved
Fu, B., Brown, C. & Mäler, L. (2020). Expression and Purification of DGD2, a Chloroplast Outer Membrane-Associated Glycosyltransferase for Galactolipid Synthesis. Biochemistry, 59(8), 999-1009
Open this publication in new window or tab >>Expression and Purification of DGD2, a Chloroplast Outer Membrane-Associated Glycosyltransferase for Galactolipid Synthesis
2020 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 59, no 8, p. 999-1009Article in journal (Refereed) Published
Abstract [en]

Galactolipids are characteristic lipids of the photosynthetic membranes. They are highly enriched in the chloroplast and are present in photosystem structures. There are two major types of galactolipids, i.e., monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) in chloroplastic membranes, which amount to similar to 50 and similar to 20 mol % of the total chloroplast lipids, respectively. Under phosphate-limiting conditions, the amount of DGDG increases dramatically for rescuing phosphate from phospholipids. In Arabidopsis thaliana, the gene digalactosyldiacylglycerol synthase 2 (DGD2) encodes a membrane-associated glycosyltransferase. The gene expression is highly responsive to phosphate starvation and is significantly upregulated in this case. To understand the molecular mechanism of DGD2, we established a protocol for DGD2 expression and purification in an Escherichia coli-based system. The work involved optimization of the expression condition and the purification protocol and a careful selection of buffer additives. It was found that a removal of around 70 C-terminal residues was necessary to produce a homogeneous monomeric protein sample with high purity, which was highly active. The purified sample was characterized by an activity assay for enzyme kinetics in which a range of membrane mimetics with different lipid compositions were used. The results demonstrate that DGD2 activity is stimulated by the presence of negatively charged lipids, which highlight the importance of the membrane environment in modulating the enzyme's activity. The study also paves way for future biophysical and structural studies of the enzyme.

Keywords
Lipids, Purification, Peptides and proteins, Genetics, Membranes
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-180606 (URN)10.1021/acs.biochem.0c00028 (DOI)000518234800010 ()32067450 (PubMedID)
Available from: 2020-04-21 Created: 2020-04-21 Last updated: 2022-02-26Bibliographically approved
Liebau, J., Tersa, M., Trastoy, B., Patrick, J., Rodrigo-Unzueta, A., Corzana, F., . . . Mäler, L. (2020). Unveiling the activation dynamics of a fold-switch bacterial glycosyltransferase by 19F NMR. Journal of Biological Chemistry, 295(29), 9868-9878
Open this publication in new window or tab >>Unveiling the activation dynamics of a fold-switch bacterial glycosyltransferase by 19F NMR
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2020 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 295, no 29, p. 9868-9878Article in journal (Refereed) Published
Abstract [en]

Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix–to–β-strand and β-strand–to–α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.

Keywords
protein structure, protein fold-switching, protein dynamics, conformational dynamics, protein function, enzyme catalysis, F-19 NMR, relaxation dispersion, carbohydrate active enzymes, glycosyltransferases
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-184456 (URN)10.1074/jbc.RA120.014162 (DOI)000553333300008 ()32434931 (PubMedID)
Available from: 2020-10-23 Created: 2020-10-23 Last updated: 2023-09-01Bibliographically approved
Liebau, J., Fu, B., Brown, C. & Mäler, L. (2018). New insights into the membrane association mechanism of the glycosyltransferase WaaG from Escherichia coli. Biochimica et Biophysica Acta - Biomembranes, 1860(3), 683-690
Open this publication in new window or tab >>New insights into the membrane association mechanism of the glycosyltransferase WaaG from Escherichia coli
2018 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 3, p. 683-690Article in journal (Refereed) Published
Abstract [en]

Monotopic glycosyltransferases (GTs) interact with membranes via electrostatic interactions. The N-terminal domain is permanently anchored to the membrane while the membrane interaction of the C-terminal domain is believed to be weaker so that it undergoes a functionally relevant conformational change upon donor or acceptor binding. Here, we studied the applicability of this model to the glycosyltransferase WaaG. WaaG is involved in the synthesis of lipopolysaccharides (LPS) in Gram-negative bacteria and was previously categorized as a monotopic GT. We analyzed the binding of WaaG to membranes by stopped-flow fluorescence and NMR diffusion experiments. We find that electrostatic interactions are required to bind WaaG to membranes while mere hydrophobic interactions are not sufficient. WaaG senses the membrane's surface charge density but there is no preferential binding to specific anionic lipids. However, the binding is weaker than expected for monotopic GTs but similar to peripheral GTs. Therefore, WaaG may be a peripheral GT and this could be of functional relevance in vivo since LPS synthesis occurs only when WaaG is membrane-bound. We could not observe a C-terminal domain movement under our experimental conditions.

Keywords
Diffusion NMR, Stopped-flow fluorescence, Vesicle, Bicelle, Membrane interaction, Lipids
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-153602 (URN)10.1016/j.bbamem.2017.12.004 (DOI)000424726800006 ()29225173 (PubMedID)
Available from: 2018-03-14 Created: 2018-03-14 Last updated: 2022-02-28Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9464-4311

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