Sequence motifs,polar interactions and conformational changes in helical membrane proteins

The alpha helices of transmembrane proteins interact to form higher order structures. These interactions are frequently mediated by packing motifs (such as GxxxG) and polar residues. Recent structural data have revealed that small sidechains are able to both stabilize helical membrane proteins and allow conformational changes in the structure. The strong interactions involving polar sidechains often contribute to protein misfolding or malfunction.     

Folding and stability of helical proteins: Carp myogen

In this work we use our very simple general representation of protein structures to study the mainly helical protein carp myogen. The representation, which treats the amino acid side-chains as simple spheres, is further simplified by rigidly fixing residues in α-helices. With this model we are able to reproduce the geometry and energetic stability of the native myogen conformation. Studies of the formation of α-helical sub-assemblies showed that the simulated folding of two and four-helix systems worked well, reaching compact native-like conformations with a good rate of success. Greater problems were encountered with the whole molecule (six helices), possibly due to the omission of entropic effects or to simulating the folding too rapidly. Finally, studies of the conformation of a pair of helices when isolated and when part of the whole molecule native conformation showed that long-range interactions have an unexpectedly strong influence on the conformation of the pair of helices.     

Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers

Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding. In addition, we find that a pair of contacting TM helices is packed tighter when there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions, including "Polar Clamp" and "Serine Zipper," where conserved Ser residues coincide with tightly packed locations in the TM region. With the examples of halorhodopsin, calcium-transporting ATPase, and bovine cytochrome c oxidase, we discuss the roles of hydrogen bonds in stabilizing helical bundles in polytopic membrane proteins and in protein functions.     

Folding and assembly of beta-barrel membrane proteins

Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins. Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro. They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes. This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes. How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed. The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer. Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro. Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.     

Experimental determination of proline hydroxylation and hydroxyproline arabinogalactosylation motifs in secretory proteins

Many secretory and several vacuolar proteins in higher plants contain hydroxylated proline residues. In many cases, hydroxyprolines in proteins are glycosylated with either arabinogalactan or oligoarabinose. We have previously shown that a sporamin precursor is O-glycosylated at the hydroxylated proline 36 residue with an arabinogalactan-type glycan when this protein is expressed in tobacco BY-2 cells (Matsuoka et al., 1995). Taking advantage of the fact that this is the only site of proline hydroxylation and glycosylation in sporamin, we analyzed the amino acid requirement for proline hydroxylation and arabinogalactosylation. We expressed several deletion constructs and many amino acid substitution mutants in tobacco cells and analyzed glycosylation and proline hydroxylation of the expressed sporamins. Hydroxylation of a proline residue requires the five amino acid sequence [AVSTG]-Pro-[AVSTGA]-[GAVPSTC]-[APS or acidic] (where Pro is the modification site) and glycosylation of hydroxyproline (Hyp) requires the seven amino acid sequence [not basic]-[not T]-[neither P, T, nor amide]-Hyp-[neither amide nor P]-[not amide]-[APST], although charged amino acids at the -2 position and basic amide residues at the +1 position relative to the modification site seem to inhibit the elongation of the arabinogalactan side chain. Based on the combination of these two requirements, we concluded that the sequence motif for efficient arabinogalactosylation, including the elongation of the glycan side chain, is [not basic]-[not T]-[AVSG]-Pro-[AVST]-[GAVPSTC]-[APS].     

Folding and stability of membrane transport proteins in vitro

Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. This review will focus on in vitro stability and folding studies of transmembrane alpha helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small number of ATP binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Folding of small helical proteins assisted by small-angle X-ray scattering profiles

This paper reports a computational method for folding small helical proteins. The goal was to determine the overall topology of proteins given secondary structure assignment on sequence. In doing so, a Monte Carlo protocol, which combines coarse-grained normal modes and a Hamiltonian at a different scale, was developed to enhance sampling. In addition to the knowledge-based potential functions, a small-angle X-ray scattering (SAXS) profile was also used as a weak constraint for guiding the folding. The algorithm can deliver structural models with overall correct topology, which makes them similar to those of 5 approximately 6 A cryo-EM density maps. The success could contribute to make the SAXS technique a fast and inexpensive solution-phase experimental method for determining the overall topology of small, soluble, but noncrystallizable, helical proteins.     

Riding the wave: structural and energetic principles of helical membrane proteins

Genome sequencing efforts have revealed that perhaps as many as 20-40% of open reading frames in complex organisms may encode proteins containing at least one helical transmembrane segment. Contrasting with this approaching tidal wave of helical membrane proteins is the fact that our understanding of the sequence-structure-function relationships for membrane proteins lags far behind that of soluble proteins. This looming reality emphasizes the tremendous biochemical and structural work that remains to be done on helical membrane proteins in order to elucidate the structural and energetic principles that specify and stabilize their folds, which define their functions. These facts are not lost on the pharmaceutical industry, where successful therapeutics and major discovery efforts are targeting membrane proteins.     

Folding and stability of helical bundle proteins from coarse‐grained models

We develop a coarse‐grained model where solvent is considered implicitly, electrostatics are included as short‐range interactions, and side‐chains are coarse‐grained to a single bead. The model depends on three main parameters: hydrophobic, electrostatic, and side‐chain hydrogen bond strength. The parameters are determined by considering three level of approximations and characterizing the folding for three selected proteins (training set). Nine additional proteins (containing up to 126 residues) as well as mutated versions (test set) are folded with the given parameters. In all folding simulations, the initial state is a random coil configuration. Besides the native state, some proteins fold into an additional state differing in the topology (structure of the helical bundle). We discuss the stability of the native states, and compare the dynamics of our model to all atom molecular dynamics simulations as well as some general properties on the interactions governing folding dynamics. Proteins 2013; 81:1200–1211. © 2013 Wiley Periodicals, Inc.     

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.     

Identification of novel sphingolipid-binding motifs in mammalian membrane proteins

Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.     

Sequence motifs and antimotifs in beta-barrel membrane proteins from a genome-wide analysis: the Ala-Tyr dichotomy and chaperone binding motifs

Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. Although sequence motifs have been studied in alpha-helical membrane proteins and have been shown to play important roles in their assembly, it is not clear whether over-represented motifs and under-represented anti-motifs exist in beta-barrel membrane proteins. We have developed probabilistic models to identify sequence motifs of residue pairs on the same strand separated by an arbitrary number of residues. A rigorous statistical model is essential for this study because of the difficulty associated with the short length of the strands and the small amount of structural data. By comparing to the null model of exhaustive permutation of residues within the same beta-strand, propensity values of sequence patterns of two residues and p-values measuring statistical significance are calculated exactly by several analytical formulae we have developed or by enumeration. We find that there are characteristic sequence motifs and antimotifs in transmembrane (TM) beta-strands. The amino acid Tyr plays an important role in several such motifs. We find a general dichotomy consisting of favorable Aliphatic-Tyr sequence motifs and unfavorable Tyr-Aliphatic antimotifs. Tyr is also part of a terminal motif, YxF, which is likely to be important for chaperone binding. Our results also suggest several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of beta-barrel membrane proteins.     

Molecular packing and packing defects in helical membrane proteins

The packing of helices spanning lipid bilayers is crucial for the stability and function of alpha-helical membrane proteins. Using a modified Voronoi procedure, we calculated packing densities for helix-helix contacts in membrane spanning domains. Our results show that the transmembrane helices of protein channels and transporters are significantly more loosely packed compared with helices in globular proteins. The observed packing deficiencies of these membrane proteins are also reflected by a higher amount of cavities at functionally important sites. The cavities positioned along the gated pores of membrane channels and transporters are noticeably lined by polar amino acids that should be exposed to the aqueous medium when the protein is in the open state. In contrast, nonpolar amino acids surround the cavities in those protein regions where large rearrangements are supposed to take place, as near the hinge regions of transporters or at restriction sites of protein channels. We presume that the observed deficiencies of helix-helix packing are essential for the helical mobility that sustains the function of many membrane protein channels and transporters.     

The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1). Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7). Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich. In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue. This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.     

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E. coli and FomA of F. nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpA is a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane beta-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded beta-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of beta-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane beta-barrels.     

Regulated shift from helical to polar localization of Listeria monocytogenes cell wall-anchored proteins

Many virulence factors of Gram-positive bacterial pathogens are covalently anchored to the peptidoglycan (PG) by sortase enzymes. However, for rod-shaped bacteria little is known about the spatiotemporal organization of these surface proteins in the cell wall. Here we report the three-dimensional (3D) localization of the PG-bound virulence factors InlA, InlH, InlJ, and SvpA in the envelope of Listeria monocytogenes under different growth conditions. We found that all PG-anchored proteins are positioned along the lateral cell wall in nonoverlapping helices. However, these surface proteins can also become localized at the pole and asymmetrically distributed when specific regulatory pathways are activated. InlA and InlJ are enriched at poles when expressed at high levels in exponential-phase bacteria. InlA and InlH, which are σ(B)dependent, specifically relocalize to the septal cell wall and subsequently to the new pole in cells entering stationary phase. The accumulation of InlA and InlH in the septal region also occurs when oxidative stress impairs bacterial growth. In contrast, the iron-dependent protein SvpA is present at the old pole and is excluded from the septum and new pole of bacteria grown under low-iron conditions. We conclude that L. monocytogenes rapidly reorganizes the spatial localization of its PG proteins in response to changes in environmental conditions such as nutrient deprivation or other stresses. This dynamic control would distribute virulence factors at specific sites during the infectious process.     

Hide and run. Arginine-based endoplasmic-reticulum-sorting motifs in the assembly of heteromultimeric membrane proteins

Arginine-based endoplasmic reticulum (ER)-localization signals are sorting motifs that are involved in the biosynthetic transport of multimeric membrane proteins. After their discovery in the invariant chain of the major histocompatibility complex class II, several hallmarks of these signals have emerged. They occur in polytopic membrane proteins that are subunits of membrane protein complexes; the presence of the signal maintains improperly assembled subunits in the ER by retention or retrieval until it is masked as a result of heteromultimeric assembly. A distinct consensus sequence and their position independence with respect to the distal termini of the protein distinguish them from other ER-sorting motifs. Recognition by the coatomer (COPI) vesicle coat explains ER retrieval. Often, di-leucine endocytic signals occur close to arginine-based signals. Recruitment of 14-3-3 family or PDZ-domain proteins can counteract ER-localization activity, as can phosphorylation. This, and the occurrence of arginine-based signals in alternatively spliced regions, implicates them in the regulated surface expression of multimeric membrane proteins in addition to their function in quality control.     

Protein-protein interactions in the membrane: sequence, structural, and biological motifs

Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.