Dipole moment in TIM alpha/beta fold proteins

TIM proteins of alpha/beta barrel fold from alpha/beta class as given in SCOP database were taken for dipole moment analysis. In all, 32 structures were analyzed for their dipole moment contributions. Representative structures from 20 super families in the alpha/beta fold, with different enzyme functions and 12 protein domains of TIM family in TIM super family were considered. The active sites of these proteins are located on the C-terminal side of the beta-strands. The molecules of same alpha/beta fold, but differing in their functionality also showed a common electrostatic field pattern along the barrel axis and had the dipole moment along the barrel axis and towards C-terminal end of the beta-strands. However, it is observed from our calculations that the dipole moment direction is possibly a consequence of the structural fold, with distribution of charges playing a modulatory role, and does not contribute to the location of active site. We show here that apart from the commonly held view as proposed by Hol et al [Hol W G L, van Duijnen PT and Berendsen H J C (1978) Nature (London), 273, 443-446] of the role of the alpha helical dipole moment, the beta-sheets in the barrel can also have a considerable dipole moment contribution. Taken together with our dipole moment analysis on integral membrane proteins [Vasanthi G and Krishnaswamy S (2002) Indian J Biochem Biophys 39, 93-100], this suggests the need to examine the role of dipole moment in the case of especially beta sheets forming barrels.     

Interaction with a lipid membrane: a key step in bacterial toxins virulence

Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane.     

Endoplasmic reticulum quality control of oligomeric membrane proteins: topogenic determinants involved in the degradation of the unassembled Na,K-ATPase alpha subunit and in its stabilization by beta subunit assembly

The molecular nature of determinants that mediate degradation of unassembled, polytopic subunits of oligomeric membrane proteins and their stabilization after partner subunit assembly is largely unknown. Expressing truncated Na,K-ATPase alpha subunits alone or together with beta subunits, we find that in unassembled alpha subunits neither the four N-terminal transmembrane segments acting as efficient alternating signal anchor-stop transfer sequences nor the large, central cytoplasmic loop exposes any degradation signal, whereas poor membrane insertion efficiency of C-terminal membrane domains M5, M7, and M9 coincides with the transient exposure of degradation signals to the cytoplasmic side. beta assembly with an alpha domain comprising at least D902 up to Y910 in the extracytoplasmic M7/M8 loop is necessary to stabilize Na,K-ATPase alpha subunits by favoring M7/M8 membrane pair formation and by protecting a degradation signal recognized from the endoplasmic reticulum (ER) lumenal side. Thus our results suggest that ER degradation of Na,K-ATPase alpha subunits is 1) mainly mediated by folding defects caused by inefficient membrane insertion of certain membrane domains, 2) a multistep process, which involves proteolytic and/or chaperone components acting from the ER lumenal side in addition to cytosolic, proteasome-related factors, and 3) prevented by partner subunit assembly because of direct protection and retrieval of degradation signals from the cytoplasm to the ER lumenal side. These results likely represent a paradigm for the ER quality control of unassembled, polytopic subunits of oligomeric membrane proteins.     

Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-State NMR

The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.     

Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins

The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel.     

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.     

Solid-state nuclear magnetic resonance studies of HIV and influenza fusion peptide orientations in membrane bilayers using stacked glass plate samples

The human immunodeficiency virus (HIV) and influenza virus fusion peptides are approximately 20-residue sequences which catalyze the fusion of viral and host cell membranes. The orientations of these peptides in lipid bilayers have been probed with 15N solid-state nuclear magnetic resonance (NMR) spectroscopy of samples containing membranes oriented between stacked glass plates. Each of the peptides adopts at least two distinct conformations in membranes (predominantly helical or beta strand) and the conformational distribution is determined in part by the membrane headgroup and cholesterol composition. In the helical conformation, the 15N spectra suggest that the influenza peptide adopts an orientation approximately parallel to the membrane surface while the HIV peptide adopts an orientation closer to the membrane bilayer normal. For the beta strand conformation, there appears to be a broader peptide orientational distribution. Overall, the data suggest that the solid-state NMR experiments can test models which correlate peptide orientation with their fusogenic function.     

A sequence-profile-based HMM for predicting and discriminating beta barrel membrane proteins

MOTIVATION: Membrane proteins are an abundant and functionally relevant subset of proteins that putatively include from about 15 up to 30% of the proteome of organisms fully sequenced. These estimates are mainly computed on the basis of sequence comparison and membrane protein prediction. It is therefore urgent to develop methods capable of selecting membrane proteins especially in the case of outer membrane proteins, barely taken into consideration when proteome wide analysis is performed. This will also help protein annotation when no homologous sequence is found in the database. Outer membrane proteins solved so far at atomic resolution interact with the external membrane of bacteria with a characteristic beta barrel structure comprising different even numbers of beta strands (beta barrel membrane proteins). In this they differ from the membrane proteins of the cytoplasmic membrane endowed with alpha helix bundles (all alpha membrane proteins) and need specialised predictors. RESULTS: We develop a HMM model, which can predict the topology of beta barrel membrane proteins using, as input, evolutionary information. The model is cyclic with 6 types of states: two for the beta strand transmembrane core, one for the beta strand cap on either side of the membrane, one for the inner loop, one for the outer loop and one for the globular domain state in the middle of each loop. The development of a specific input for HMM based on multiple sequence alignment is novel. The accuracy per residue of the model is 83% when a jack knife procedure is adopted. With a model optimisation method using a dynamic programming algorithm seven topological models out of the twelve proteins included in the testing set are also correctly predicted. When used as a discriminator, the model is rather selective. At a fixed probability value, it retains 84% of a non-redundant set comprising 145 sequences of well-annotated outer membrane proteins. Concomitantly, it correctly rejects 90% of a set of globular proteins including about 1200 chains with low sequence identity (<30%) and 90% of a set of all alpha membrane proteins, including 188 chains.     

Dissection of β‐barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero‐oligomeric machinery that mediates β‐barrel outer membrane protein (OMP) assembly. The C‐ and N‐termini of BamA fold into trans‐membrane β‐barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β‐barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site‐specific cross‐linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β‐barrel OMPs except for TolC, which folds into a unique soluble α‐helical barrel and an OM‐anchored β‐barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans‐membrane β‐barrel domain of BamA.     

Requirement for YaeT in the outer membrane assembly of autotransporter proteins

Autotransporters constitute the largest group of secreted proteins in gram-negative bacteria. Autotransporter secretion involves the insertion of a carboxy-terminal beta barrel into and the translocation of an amino-terminal domain across the outer membrane. Here, we demonstrate that secretion of autotransporters from several organisms requires the outer membrane assembly factor YaeT.     

Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Here we report the crystal structure of Skp from E. coli. The structure of the Skp trimer resembles a jellyfish with alpha-helical tentacles protruding from a beta barrel body defining a central cavity. The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC, a cytosolic chaperone present in eukaria and archea, that binds unfolded substrates in its central cavity. The ability of Skp to prevent the aggregation of model substrates in vitro is independent of ATP. Skp can interact directly with membrane lipids and lipopolysaccharide (LPS). These interactions are needed for efficient Skp-assisted folding of membrane proteins. We have identified a putative LPS binding site on the outer surface of Skp and propose a model for unfolded substrate binding.     

Neutron diffraction reveals sequence-specific membrane insertion of pre-fibrillar islet amyloid polypeptide and inhibition by rifampicin

Human islet amyloid polypeptide (hIAPP) forms amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM). Pre-fibrillar hIAPP oligomers (in contrast to monomeric IAPP or mature fibrils) increase membrane permeability, suggesting an important role in the disease. In the first structural study of membrane-associated hIAPP, lamellar neutron diffraction shows that oligomeric hIAPP inserts into phospholipid bilayers, and extends across the membrane. Rifampicin, which inhibits hIAPP-induced membrane permeabilisation in functional studies, prevents membrane insertion. In contrast, rat IAPP (84% identical to hIAPP, but non-amyloidogenic) does not insert into bilayers. Our findings are consistent with the hypothesis that membrane-active pre-fibrillar hIAPP oligomers insert into beta cell membranes in NIDDM.     

Energetic approach to the folding of alpha/beta barrels

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition

Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Conformational Changes in BAK,a Pore-forming Proapoptotic Bcl-2 Family Member,upon Membrane Insertion and Direct Evidence for the Existence of BH3-BH3 Contact Interface in BAK Homo-oligomers

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni²⁺-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the α5-α6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5–10 Å distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.     

Beta barrel trans-membrane proteins: Enhanced prediction using a Bayesian approach

Membrane proteins, which constitute approximately 20% of most genomes, form two main classes: alpha helical and beta barrel transmembrane proteins. Using methods based on Bayesian Networks, a powerful approach for statistical inference, we have sought to address beta-barrel topology prediction. The beta-barrel topology predictor reports individual strand accuracies of 88.6%. The method outlined here represents a potentially important advance in the computational determination of membrane protein topology.     

Oligomeric structure, dynamics, and orientation of membrane proteins from solid-state NMR

Solid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both alpha-helical and beta sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The beta-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D2O for days and is assigned to the transmembrane beta-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D2O are assigned to the extracellular and periplasmic loops. The two-dimensional 1H/15N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5 degrees tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to alpha-helical membrane proteins.