Disruption of a specific molecular interaction with a bound lipid affects the thermal stability of the purple bacterial reaction centre

Relatively little is known about the functions of specific molecular interactions between membrane proteins and membrane lipids. The structural and functional consequences of disrupting a previously identified interaction between a molecule of the diacidic lipid cardiolipin and the purple bacterial reaction centre were examined. Mutagenesis of a highly conserved arginine (M267) that is responsible for binding the head-group of the cardiolipin (to leucine) did not affect the rate of photosynthetic growth, the functional properties of the reaction centre, or the X-ray crystal structure of the complex (determined to a resolution of 2.8 A). However, the thermal stability of the protein was compromised by this mutation, part of the reaction centre population showing an approximately 5 degrees C decrease in melting temperature in response to the arginine to leucine mutation. The crystallised mutant reaction centre also no longer bound detectable amounts of cardiolipin at this site. Taken together, these observations suggest that this particular protein-lipid interaction contributes to the thermal stability of the complex, at least when in detergent micelles. These findings are discussed in the light of proposals concerning the unfolding processes that occur when membrane proteins are heated, and we propose that one function of the cardiolipin is to stabilise the interaction between adjacent membrane-spanning alpha-helices in a region where there are no direct protein-protein interactions.     

On the Role of Basic Residues in Adapting the Reaction Centre–LH1 Complex for Growth at Elevated Temperatures in Purple Bacteria

The purple photosynthetic bacterium Thermochromatium tepidum is a moderate thermophile, with a growth optimum of 48–50 °C. The X-ray crystal structure of the reaction centre from this organism has been determined, and compared with that from mesophilic bacteria such as Blastochloris viridis and Rhodobacter sphaeroides (Nogi T et al. (2000) Proc Natl Acad Sci USA 97: 13561–13566). Structural features that could contribute to the enhanced thermal stability of the Thermochromatium tepidum reaction centre were discussed, including three arginine residues exposed at the periplasmic side of the membrane that are not present in reaction centres from mesophilic organisms, and potentially could increase the affinity of the complex for the surrounding membrane. In the present report these arginine residues, plus a histidine identified from an extensive sequence alignment, were engineered into structurally homologous positions in the Rhodobacter sphaeroides reaction centre, and the effect on the thermal stability of the Rhodobacter sphaeroides complex was examined. We find that these residues do not enhance the thermal stability of the reaction centre, as assessed by absorbance spectroscopy of the bacteriochlorin cofactors in membrane-bound reaction centres. Possible roles of these residues in the Thermochromatium tepidum reaction centre are discussed, and it is proposed that they facilitate stronger binding of the reaction centre to the encircling LH1 antenna complex, through ionic interactions with acidic residues at the C-terminal end of the LH1 α-polypeptide. Such an interaction could enhance the stability of the so-called ‘RC–LH1 core’ complex that is formed between the reaction centre and the LH1 antenna, and which represents the minimal functional photosynthetic unit in all known purple photosynthetic bacteria. Stronger bonding interactions between the two complexes could also contribute to an increase in the rigidity of the photosynthetic membrane in Thermochromatium tepidum, in accord with the general finding that the cytoplasmic membrane from thermophilic eubacteria is less fluid than its counterpart in mesophilic bacteria.     

Opposing structural changes in two symmetrical polypeptides bring about opposing changes to the thermal stability of a complex integral membrane protein

The relationship between membrane protein structure and thermal stability has been examined in the reaction centre from the bacterium Rhodobacter sphaeroides, a complex membrane protein comprising three polypeptide chains and 10 cofactors. The core of this protein exhibits an approximate twofold symmetry, the cofactors being held in two membrane-spanning branches by two polypeptides, termed L and M, that have very similar folds. In assays of the thermal stability of wild-type and mutant reaction centres embedded in the native bilayer membrane, replacement of a Phe at position 197 of the M polypeptide by His produced an increase in stability, whereas an opposing replacement of His by Phe at the symmetrical position 168 of the L-polypeptide produced a decrease in stability. In light of the known X-ray crystal structures of wild-type and mutant variants of this protein, and further mutagenesis, it is concluded that these stability changes result from the introduction or removal, respectively, of a hydrogen bond between the side-chain of the His and that of an Asn located two positions along the M or L polypeptide chain, in addition to a hydrogen bond between the His side-chain and an adjacent bacteriochlorophyll cofactor.     

Mitochondrial kinases and their molecular interaction with cardiolipin

Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) are not phylogenetically related but share functionally important properties. They both use mitochondrially generated ATP with the ultimate goal of maintaining proper nucleotide pools, are located in the intermembrane/cristae space, have symmetrical oligomeric structures, and show high affinity binding to anionic phospholipids, in particular cardiolipin. The structural basis and functional consequences of the cardiolipin interaction have been studied and are discussed in detail in this review. They mainly result in a functional interaction of MtCK and NDPK-D with inner membrane adenylate translocator, probably by forming proteolipid complexes. These interactions allow for privileged exchange of metabolites (channeling) that ultimately regulate mitochondrial respiration. Further functions of the MtCK/membrane interaction include formation of cardiolipin membrane patches, stabilization of mitochondria and a role in apoptotic signaling, as well as in case of both kinases, a role in facilitating lipid transfer between two membranes. Finally, disturbed cardiolipin interactions of MtCK, NDPK-D and other proteins like cytochrome c and truncated Bid are discussed more generally in the context of apoptosis and necrosis.     

A network representation of protein structures: implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.     

Dystrophin: More than just the sum of its parts

Dystrophin is one of a number of large cytoskeleton associated proteins that connect between various cytoskeletal elements and often are tethered to the membrane through other transmembrane protein complexes. These cytolinker proteins often provide structure and support to the cells where they are expressed, and mutations in genes encoding these proteins frequently gives rise to disease. Dystrophin is no exception in any of these respects, providing connections between a transmembrane complex known as the dystrophin–glycoprotein complex and the underlying cytoskeleton. The most established connection and possibly the most important is that to F-actin, but more recently evidence has been forthcoming of connections to membrane phospholipids, intermediate filaments and microtubules. Moreover it is becoming increasingly clear that the multiple spectrin-like repeats in the centre of the molecule, that had hitherto been thought to be largely redundant, harbour binding activities that have a significant impact on dystrophin functionality. This functionality is particularly apparent when assessed by the ability to rescue the dystrophic phenotype in mdx mice. This review will focus on the relatively neglected but functionally vital coiled-coil region of dystrophin, highlighting the structural relationships and interactions of the coiled-coil region and providing new insights into the functional role of this region.     

Identification of a Novel Role for Dematin in Regulating Red Cell Membrane Function by Modulating Spectrin-Actin Interaction

The membrane skeleton plays a central role in maintaining the elasticity and stability of the erythrocyte membrane, two biophysical features critical for optimal functioning and survival of red cells. Many constituent proteins of the membrane skeleton are phosphorylated by various kinases, and phosphorylation of β-spectrin by casein kinase and of protein 4.1R by PKC has been documented to modulate erythrocyte membrane mechanical stability. In this study, we show that activation of endogenous PKA by cAMP decreases membrane mechanical stability and that this effect is mediated primarily by phosphorylation of dematin. Co-sedimentation assay showed that dematin facilitated interaction between spectrin and F-actin, and phosphorylation of dematin by PKA markedly diminished this activity. Quartz crystal microbalance measurement revealed that purified dematin specifically bound the tail region of the spectrin dimer in a saturable manner with a submicromolar affinity. Pulldown assay using recombinant spectrin fragments showed that dematin, but not phospho-dematin, bound to the tail region of the spectrin dimer. These findings imply that dematin contributes to the maintenance of erythrocyte membrane mechanical stability by facilitating spectrin-actin interaction and that phosphorylation of dematin by PKA can modulate these effects. In this study, we have uncovered a novel functional role for dematin in regulating erythrocyte membrane function.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm

The TonB protein is required to transduce energy from the cytoplasmic membrane to outer membrane transport proteins of Gram-negative bacteria. Two accessory proteins, ExbB and ExbD, are required for TonB function and it has been suggested that TonB and ExbBD form a complex in the membrane. In this paper we demonstrate that there are two spatially distinct, functional interactions between ExbBD and TonB. First, there is an interaction between ExbBD and the N-terminal signal-like peptide of TonB, probabiy the formation of a stable complex in the membrane. Second, ExbB interacts with TonB in the cytoplasm. This interaction involves the domain of TonB that is normally periplasmic. Thus, this is a transient interaction which occurs during the synthesis and/or localization of TonB, implying a chaperone-like role for ExbB. The transmembrane topology of ExbB was shown to be consistent with this role.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.     

The lipid environment of Escherichia coli Aquaporin Z

In this study, we have investigated the lipids surrounding AqpZ, and the effects of a destabilizing mutation W₁₄A (Schmidt and Sturgis, 2017) on lipid protein interactions. In a first approach, we used Styrene Maleic Acid copolymer to prepare AqpZ containing nanodiscs, and these were analyzed for their lipid content, investigating both the lipid head-group and acyl-chain compositions. These results were complemented by native mass spectrometry of purified AqpZ in the presence of lipids, to give insights of variations in lipid binding at the surface of AqpZ. In an effort to gain molecular insights, to aid interpretation of these results, we performed a series of coarse grained molecular dynamics simulations of AqpZ, in mixed lipid membranes, and correlated our observations with the experimental measurements. These various results are then integrated to give a clearer picture of the lipid environment of AqpZ, both in the native membrane, and in lipid nanodiscs. We conclude that AqpZ contains a lipid binding-site, at the interface between the monomers of the tetramer, that is specific for cardiolipin. Almost all the cardiolipin, in AqpZ containing nanodiscs, is probably associated with this site. The SMA 3:1 nanodiscs we obtained contain a rather high proportion of lipid, and in the case of nanodiscs containing AqpZ cardiolipin is depleted. This is possibly because, in the membrane, there is little cardiolipin not associated with binding sites on the surface of the different membrane proteins. Surprisingly, we see no evidence for lipid sorting based on acyl chain length, even in the presence of a large hydrophobic mismatch, suggesting that conformational restrictions are energetically less costly than lipid sorting.     

Vesicle Fusion Probability Is Determined by the Specific Interactions of Munc18

Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes. Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Disruption of munc18 binding to the N-terminal peptide motif of syntaxin did not alter munc18 localization on the plasma membrane but had a pronounced influence on the behavior of secretory vesicles and their likelihood to undergo fusion. We therefore conclude that interaction with the syntaxin N-peptide can confer differential release probabilities to secretory vesicles and may contribute to the delineation of secretory vesicle pools.     

Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.     

Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins

The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes.     

Yet1p and Yet3p,the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1Δ and yet3Δ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1Δ or yet3Δ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.     

Improvement of model for prediction of hemagglutinin mutations in H5N1 influenza viruses with distinguishing of arginine, leucine and serine

In a continuation of our attempt to predict mutations in proteins from influenza A virus, this study attempted to answer the question of whether distinguishing between arginine, leucine and serine can improve the predictability as these residues are governed by different probabilistic mechanism translating from RNA codons to amino acids. In this study, we made the prediction based on the mutation relation among 299 H5N1 hemagglutinins of influenza A virus. Then, we compared the results based on the distinguishing of arginine, leucine and serine with the results without distinguishing of arginine, leucine and serine. The results show that the prediction together with distinguishing between arginine, leucine and serine is better than prediction without distinguishing between these residues.     

The potassium channel KcsA: a model protein in studying membrane protein oligomerization and stability of oligomeric assembly?

Many membrane proteins are functional as stable oligomers. An understanding of the conditions that elicit and enhance oligomerization is important in many therapeutics. In this regard, protein–protein and protein–lipid interactions play crucial roles in the assembly and stability of oligomeric complexes. Recent years have seen a rapid increase in the mechanistic information on the importance of cytoplasmic termini in determining subunit assembly and stability of oligomeric complexes. In addition, the role of specific protein–lipid interaction between anionic phospholipids and “hot spots” on the protein surface has also become evident in stabilizing oligomeric assemblies. This review focuses on several contemporary developments of membrane proteins that stabilize oligomers by taking the potassium channel KcsA as an exemplary ion channel.     

YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.     

Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability

Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the interactions between the different proteins, we used the two-hybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement. To define the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1. The putative alpha helix present in the MADS-box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII. Interactions between the two MADS-box proteins and ArgRIII were confirmed using affinity chromatography. The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the 'arginine boxes'. We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain.     

Cardiolipin dynamics and binding to conserved residues in the mitochondrial ADP/ATP carrier

Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids—with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.