Complete topology inversion can be part of normal membrane protein biogenesis

The topology of helical membrane proteins is generally defined during insertion of the transmembrane helices, yet it is now clear that it is possible for topology to change under unusual circumstances. It remains unclear, however, if topology reorientation is part of normal biogenesis. For dual topology dimer proteins such as the multidrug transporter EmrE, there may be evolutionary pressure to allow topology flipping so that the populations of both orientations can be equalized. We previously demonstrated that when EmrE is forced to insert in a distorted topology, topology flipping of the first transmembrane helix can occur during translation. Here, we show that topological malleability also extends to the C‐terminal helix and that even complete topology inversion of the entire EmrE protein can occur after the full protein is translated and inserted. Thus, topology rearrangements are possible during normal biogenesis. Wholesale topology flipping is remarkable given the physical constraints of the membrane and expands the range of possible membrane protein folding pathways, both productive and detrimental.     

Quasi-symmetry in the cryo-EM structure of EmrE provides the key to modeling its transmembrane domain

Small multidrug resistance (SMR) transporters contribute to bacterial resistance by coupling the efflux of a wide range of toxic aromatic cations, some of which are commonly used as antibiotics and antiseptics, to proton influx. EmrE is a prototypical small multidrug resistance transporter comprising four transmembrane segments (M1-M4) that forms dimers. It was suggested recently that EmrE molecules in the dimer have different topologies, i.e. monomers have opposite orientations with respect to the membrane plane. A 3-D structure of EmrE acquired by electron cryo-microscopy (cryo-EM) at 7.5 Angstroms resolution in the membrane plane showed that parts of the structure are related by quasi-symmetry. We used this symmetry relationship, combined with sequence conservation data, to assign the transmembrane segments in EmrE to the densities seen in the cryo-EM structure. A C alpha model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix. The model is validated by much of the biochemical data on EmrE with most of the positions that were identified as affecting substrate translocation being located around the substrate-binding cavity. A suggested mechanism for proton-coupled substrate translocation in small multidrug resistance antiporters provides a mechanistic rationale to the experimentally observed inverted topology.     

Convergence of experimental, computational and evolutionary approaches predicts the presence of a tetrameric form for CD3-zeta

Experimental results using multiple site-specific infrared dichroism have shown that, when reconstituted into lipid bilayers, the orientation of the transmembrane domain of CD3-zeta is not compatible with a dimeric right-handed model reported previously. This model, obtained using a computational approach that uses evolutionary data, is in agreement with mutagenesis data and homology modelling. This suggested that, in our experimental conditions, the oligomeric state of CD3-zeta may not be dimeric. We have explored this possibility by performing global searching molecular dynamics simulations assuming different homo-oligomeric sizes (from 2 to 6). In these simulations, the helix tilt was restrained to the average helix tilt obtained experimentally, 12 degrees. Only a left-handed tetrameric model was compatible with the experimentally observed tilt and rotational orientation of the helix, and was also the lowest-energy model amongst the candidate structures obtained. Furthermore, simulations performed using close homologues demonstrate that this model is compatible with evolutionary conservation data. Finally, the pattern of residue conservation in the zeta family of proteins strongly argues in favour of the presence of a left-handed hetero-oligomer with an orientation compatible with the tetramer we present. These results show that both the known dimeric and the so far undetected tetrameric form may be of functional importance in the cell.     

Structural comparison and classification of alpha‐helical transmembrane domains based on helix interaction patterns

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three‐dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies α‐helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of α‐helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Anti-apoptotic Bcl-XL protein in complex with BH3 peptides of pro-apoptotic Bak, Bad, and Bim proteins: comparative molecular dynamics simulations

The Bcl-2 family of proteins plays a central role in the regulation of mitochondrial outer-membrane permeabilization, a critical step in apoptosis. Heterodimerization between the pro- and anti-apoptotic members of Bcl-2 family is a key event in this process. Anti-apoptotic proteins have high levels of expression in many cancers and they have different affinities for different pro-apoptotic proteins. Experimentally determined structures of all members of Bcl-2 proteins have remarkably similar helical fold despite poor amino acid sequence identity. Peptides representing BH3 region of pro-apoptotic proteins have been shown to bind the hydrophobic cleft of anti-apoptotic proteins and this segment is responsible in modulating the apoptotic pathways in living cells. Understanding the molecular basis of protein-protein recognition is required to develop inhibitors specific to a particular anti-apoptotic protein. We have carried out molecular dynamics simulations on the anti-apoptotic Bcl-X(L) protein in complex with three different BH3 peptides derived from pro-apoptotic Bak, Bad and Bim proteins. Each complex structure was simulated for a period of 50 ns after 2.5 ns equilibration. Analysis of the simulation results showed that in the Bcl-X(L) protein, the helix containing the BH3 region is more flexible than other helices in all three simulations. A network of strong hydrophobic interactions exists between four of the six helices and they contribute significantly to the stability of this helix bundle protein. Analysis of Bcl-X(L)-BH3 peptide interactions reveals the role of loop residues in the protein-peptide interactions in all three simulations. Bad and Bim peptides maintain strong hydrophobic and hydrophilic interactions with the helix preceding the central hydrophobic helix. Residues from this helix interact with an Arg residue in Bad and Bim peptides. This Arg residue is next to the conserved Leu residue and is replaced by Ala in Bak. Absence of these interactions and the helix propensity are likely to be the cause for Bak peptide's weaker binding affinity with the Bcl-X(L) protein. The results of this study have implications in the design of Bcl-X(L)-specific inhibitors.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis

Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.     

Structural characterization by computer experiments of the lipid-free LDL-receptor-binding domain of apolipoprotein E.

The structure and dynamics of the lipid-free LDL-receptor-binding domain of apolipoprotein E (apoE-RBD) has been investigated by Molecular Dynamics Simulations. ApoE-RBD in its monomeric lipid-free form is a singular four-helix bundle made up of four elongated amphipathic helices. Analysis of one 1.5 ns molecular dynamics trajectory of apoE-RBD performed in water indicates that the lipid-free domain adopts a structure that exhibits characteristics found in native proteins: it has very stable helices and presents a compact structure. Yet its interior exhibits a larger number of transient atomic-size cavities relative to that found in other proteins of similar size and its apolar side chains are more mobile. The latter features distinguish the elongated four-helix bundle as a slightly disordered structure, which shows a structural likeness with some de novo designed four-helix bundle proteins and shares with the latter a leucine-rich residue composition. We anticipate that these unique properties compared with other native helix bundles may be related to the postulated ability of apoE-RBD to undergo an opening of its bundle upon interaction with phospholipids. The distribution of empty cavities computed along the trajectory in the interface regions between the different pairs of helices reveals that the tertiary contacts in one of the interfaces are weaker suggesting that this particular interface could be more easily ruptured upon lipid association.     

Structure of the substrate binding pocket of the multidrug transporter EmrE: site-directed spin labeling of transmembrane segment 1

Site-directed spin labeling (SDSL) was used to explore the structural framework responsible for the obligatory drug-proton exchange in the Escherichia coli multidrug transporter, EmrE. For this purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS1). This segment has been implicated in the catalytic mechanism of EmrE due to the presence of the highly conserved glutamate 14, a residue absolutely required for ligand binding. Sequence-specific variation in the accessibilities of the introduced nitroxides to molecular oxygen reveals a transmembrane helical conformation along TMS1. One face of the helix is in contact with the hydrocarbon interior of the detergent micelle while the other face appears to be solvated by an aqueous environment, resulting in significant exposure of the nitroxides along this face to NiEDDA. TMS1 from two different subunits are in close proximity near a 2-fold axis of symmetry as revealed by the analysis of spin-spin interactions at sites 14 and 18. The limited extent of spin-spin interactions is consistent with a scissor-like packing of the two TMS1. This results in a V-shaped chamber which is in contact with the aqueous phase near the N-terminus. The spatial organization of TMS1, particularly the close proximity of E14, is consistent with a proposed mechanistic model of EmrE [Yerushalmi, H., and Schuldiner, S. (2000) Biochemistry 39, 14711-14719] where substrate extrusion is coupled to proton influx through electrostatic interactions and shifts of the glutamate 14 pK(a) during the cycle.     

A model for the coupling of alpha-helix and tertiary contact formation

Peptides corresponding to excised alpha-helical segments of natural proteins can spontaneously form helices in solution. However, peptide helices are usually substantially less stable in solution than in the structural context of a folded protein, because of the additional interactions possible between helices in a protein. Such interactions can be thought of as coupling helix formation and tertiary contact formation. The relative energetic contributions of the two processes to the total energy of the folded state of a protein is a matter of current debate. To investigate this balance, an extended helix-coil model (XHC) that incorporates both effects has been constructed. The model treats helix formation with the Lifson-Roig formalism, which describes helix initiation and propagation through cooperative local interactions. The model postulates an additional parameter representing participation of a site in a tertiary contact. In the model, greater helix stability can be achieved through combinations of these short-range and long-range interactions. For instance, stronger tertiary contacts can compensate for helices with little intrinsic stability. By varying the strength of the nonlocal interactions, the model can exhibit behavior consistent with a variety of qualitative models describing the relative importance of secondary and tertiary structure. Moreover, the model is explicit in that it can be used to fit experimental data to individual peptide sequences, providing a means to quantify the two contributions on a common energetic basis.     

Structure of the influenza C virus CM2 protein transmembrane domain obtained by site-specific infrared dichroism and global molecular dynamics searching

The 115-residue protein CM2 from Influenza C virus has been recently characterized as a tetrameric integral membrane glycoprotein. Infrared spectroscopy and site-directed infrared dichroism were utilized here to determine its transmembrane structure. The transmembrane domain of CM2 is alpha-helical, and the helices are tilted by beta = (14.6 +/- 3.0) degrees from the membrane normal. The rotational pitch angle about the helix axis omega for the 1-(13)C-labeled residues Gly(59) and Leu(66) is omega = (218 +/- 17) degrees, where omega is defined as zero for a residue pointing in the direction of the helix tilt. A detailed structure was obtained from a global molecular dynamics search utilizing the orientational data as an energy refinement term. The structure consists of a left-handed coiled-coil with a helix crossing angle of Omega = 16 degrees. The putative transmembrane pore is occluded by the residue Met(65). In addition hydrogen/deuterium exchange experiments show that the core is not accessible to water.     

Transition-states in protein folding kinetics: the structural interpretation of Phi values

Phi values are experimental measures of the effects of mutations on the folding kinetics of a protein. A central question is what structural information Phi values give about the transition-state of folding. Traditionally, a Phi value is interpreted as representing the "nativeness" of a mutated residue in the transition-state. However, this interpretation is often problematic. We present here a better structural interpretation of Phi values for mutations within a given helix. Our interpretation is based on a simple physical model that distinguishes between secondary and tertiary free energy contributions of helical residues. From a linear fit of the model to experimental data, we obtain two structural parameters: the extent of helix formation in the transition-state, and the nativeness of tertiary interactions in the transition-state. We apply the model to all proteins with well-characterized helices for which more than 10 Phi values are available: protein A, CI2, and protein L. The model is simple to apply to experimental data, captures nonclassical Phi values <0 or >1 in these helices, and explains how different mutations at a given site can lead to different Phi values.     

Folding elastic transmembrane helices to fit in a low-resolution image by electron microscopy

Structure prediction of membrane proteins could be constrained and thereby improved by introducing data of the observed molecular shape. We studied a coarse-grained molecular model that relied on residue-based dummy atoms to fold the transmembrane helices of a protein in the observed molecular shape. Based on the inter-residue potential, the α-helices were folded to contact each other in a simulated annealing protocol to search optimized conformation. Fitting the model into a three-dimensional volume was tested for proteins with known structures and resulted in a fairly reasonable arrangement of helices. In addition, the constraint to the packing transmembrane helix with the two-dimensional region was tested and found to work as a very similar folding guide. The obtained models nicely represented α-helices with the desired slight bend. Our structure prediction method for membrane proteins well demonstrated reasonable folding results using a low-resolution structural constraint introduced from recent cell-surface imaging techniques.     

Structural investigation of helices II, III, and IV of B. megaterium 5S ribosomal RNA by molecular dynamics calculations.

The structures of the helices II-III region and the helix IV region of B. megaterium 5S rRNA have been examined by means of energy minimization and molecular dynamics calculations. Calculated distances between neighboring hydrogen-bonded imino protons in helices II, III, and IV were between 3.5 and 4.5 A. The overall axis for the helices II-III region is warped rather than straight. Formation of additional Watson-Crick base pairs in loop B and loop C was not evident from the atomic positions calculated by molecular dynamics. Bases in loop C are well stacked, showing no significant change during dynamics. Bulge migration in helix III does not seem to be possible; the helices II-III region prefers one conformation. Helix II is more stable than helix III. Five base pairs in helix IV were sufficiently stable to establish that helix IV is terminated by a hairpin loop of three nucleotides. U87 protrudes from loop D. Structures of the helices II-III segment and the helix IV segment of B. megaterium 5S rRNA obtained by molecular dynamics were generally consistent with the solution structure inferred from high-field proton nmr spectroscopy.     

Coarse-grained simulations of conformational dynamics of proteins: Application to apomyoglobin

A coarse-grained dynamic Monte Carlo method is proposed for investigating the conformational dynamics of proteins. Each residue is represented by two interaction sites, one at the α-carbon, and the other on the amino acid sidechain. Geometry and energy parameters extracted from databank structures are used. The method is applied to the crystal structure of apomyoglobin (apo-Mb). Equilibrium and dynamic properties of apo-Mb are characterized within computation times one order of magnitude shorter than conventional molecular dynamics (MD) simulations. The calculated rms fluctuations in α-carbons are in good agreement with crystallographic temperature factors. Regions exhibiting enhanced conformational mobilities are identified. Among the loops connecting the eight helices A to H, the loop CD undergoes the fastest motions, leading to partial unwinding of helix D. Helix G is the most stable helix on the basis of the kinetic stability of dihedral angles, followed by the respective helices A, E, H, and B. These results, in agreement with H/D exchange and two-dimensional NMR experiments, as well as with MD simulations, lend support to the use of the proposed approach as an efficient, yet physically plausible, means of characterizing protein conformational dynamics. Proteins 31:271–281, 1998. © 1998 Wiley-Liss, Inc.     

Robust Driving Forces for Transmembrane Helix Packing

The packing structures of transmembrane helices are traditionally attributed to patterns in residues along the contact surface. In this view, besides keeping the helices confined in the membrane, the bilayer has only a minor effect on the helices structure. Here, we use two different approaches to show that the lipid environment has a crucial effect in determining the cross-angle distribution of packed helices. We analyzed structural data of a membrane proteins database. We show that the distribution of cross angles of helix pairs in this database is statistically indistinguishable from the cross-angle distribution of two noninteracting helices imbedded in the membrane. These results suggest that the cross angle is, to a large extent, determined by the tilt angle of the individual helices. We test this hypothesis using molecular simulations of a coarse-grained model that contains no specific residue interactions. These simulations reproduce the same cross-angle distribution as found in the database. As the tilt angle of a helix is dominated by hydrophobic mismatch between the protein and surrounding lipids, our results indicate that hydrophobic mismatch is the dominant factor guiding the transmembrane helix packing. Other short-range forces might then fine-tune the structure to its final configuration.     

Structural analysis of synthetic peptide fragments from EmrE,a multidrug resistance protein,in a membrane-mimetic environment

EmrE, a multidrug resistance protein from Escherichia coli, renders the bacterium resistant to a variety of cytotoxic drugs by active translocation out of the cell. The 110-residue sequence of EmrE limits the number of structural possibilities that can be envisioned for this membrane protein. Four helix bundle models have been considered [Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1996) J. Biol. Chem. 271, 31044-31048]. The validity of EmrE structural models has been probed experimentally by investigations on overlapping peptides (ranging in length from 19 to 27 residues), derived from the sequence of EmrE. The choice of peptides was made to provide sequences of two complete, predicted transmembrane helices (peptides H1 and H3) and two helix-loop-helix motifs (peptides A and B). Peptide (B) also corresponds to a putative hairpin in a speculative beta-barrel model, with the "Pro-Thr-Gly" segment forming a turn. Structure determination in SDS micelles using NMR indicates peptide H1 to be predominantly helical, with helix boundaries in the micellar environment corroborating predicted helical limits. Peptide A adopts a helix-loop-helix structure in SDS micelles, and peptide B was also largely helical in micellar environments. An analogue peptide, C, in which the central "Pro-Thr-Gly" was replaced by "(D)Pro-Gly" displays local turn conformation at the (D)Pro-Gly segment, but neither a continuous helical stretch nor beta-hairpin formation was observed. This study implies that the constraints of membrane and micellar environments largely direct the structure of transmembrane peptides and proteins and study of judiciously selected peptide fragments can prove useful in the structural elucidation of membrane proteins.     

The Folding Transition-State Ensemble of a Four-Helix Bundle Protein: Helix Propensity as a Determinant and Macromolecular Crowding as a Probe

The four-helix bundle protein Rd-apocyt b₅₆₂, a redesigned stable variant of apocytochrome b₅₆₂, exhibits two-state folding kinetics. Its transition-state ensemble has been characterized by Φ-value analysis. To elucidate the molecular basis of the transition-state ensemble, we have carried out high-temperature molecular dynamics simulations of the unfolding process. In six parallel simulations, unfolding started with the melting of helix I and the C-terminal half of helix IV, and followed by helix III, the N-terminal half of helix IV and helix II. This ordered melting of the helices is consistent with the conclusion from native-state hydrogen exchange, and can be rationalized by differences in intrinsic helix propensity. Guided by experimental Φ-values, a putative transition-state ensemble was extracted from the simulations. The residue helical probabilities of this transition-state ensemble show good correlation with the Φ-values. To further validate the putative transition-state ensemble, the effect of macromolecular crowding on the relative stability between the unfolded ensemble and the transition-state ensemble was calculated. The resulting effect of crowding on the folding kinetics agrees well with experimental observations. This study shows that molecular dynamics simulations combined with calculation of crowding effects provide an avenue for characterize the transition-state ensemble in atomic details.     

Transmembrane helix dimerization: beyond the search for sequence motifs

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.