Galactolipids are characteristic lipids of the photosynthesis membranes of higher plants and cyanobacteria. Due to their close relationship to the stability of the photosystem protein complexes, the biogenesis of galactolipids has been intensively studied on the genetic and molecular levels. There are two major types of galactolipids in chloroplastic membranes: monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG). Under phosphate-limiting conditions, the amount of DGDG increases dramatically to allow for phosphate salvage from phospholipids. In Arabidopsis thaliana, the membrane-associated glycosyltransferase digalactosyldiacylglycerol synthase 2 (atDGD2) is highly responsive to phosphate starvation and is significantly upregulated during such conditions. The lipid galactosylation reactions are also fundamentally interesting as they require a catalyst that is capable of bringing a hydrophilic and lipophilic substrate together at the solution-membrane phase border. Here, we present the X-ray crystal structure of atDGD2, which is the first reported DGDG synthase structure. AtDGD2 is most structurally similar to functionally unrelated GT-B enzymes. Interestingly, in spite of significant donor substrate binding differences, we identified four amino acids (Gly22, His151, Lys243, and Glu321, atDGD2 numbering) which were entirely conserved between the structurally similar enzymes. We also investigated the membrane interaction kinetics and membrane anchoring mechanism of atDGD2. This demonstrated that atDGD2 is membrane-bound but also showed that membrane binding is highly dynamic. Furthermore, our structural information in context of previous biophysical studies highlights regions of the enzyme exhibiting a high degree of structural plasticity, which we propose to be important for allowing atDGD2 to quickly adapt its activity based on the membrane lipid environment.

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.

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in the synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein, we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose, and UDP-GalNAc. Structural comparison with the homolog from Escherichia coli (EcWaaG) revealed five key differences in the sugar-binding pocket. Solution-state NMR analysis showed that WT PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither WT PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

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. 

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.

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.

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.

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.

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.

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.