Insights into correlated motions and long-range interactions in CheY derived from molecular dynamics simulations

CheY is a response regulator protein involved in bacterial chemotaxis. Much is known about its active and inactive conformations, but little is known about the mechanisms underlying long-range interactions or correlated motions. To investigate these events, molecular dynamics simulations were performed on the unphosphorylated, inactive structure from Salmonella typhimurium and the CheY-BeF(3)(-) active mimic structure (with BeF(3)(-) removed) from Escherichia coli. Simulations utilized both sequences in each conformation to discriminate sequence- and structure-specific behavior. The previously identified conformational differences between the inactive and active conformations of the strand-4-helix-4 loop, which are present in these simulations, arise from the structural, and not the sequence, differences. The simulations identify previously unreported structure-specific flexibility features in this loop and sequence-specific flexibility features in other regions of the protein. Both structure- and sequence-specific long-range interactions are observed in the active and inactive ensembles. In the inactive ensemble, two distinct mechanisms based on Thr-87 or Ile-95 rotameric forms, are observed for the previously identified g+ and g- rotamer sampling by Tyr-106. These molecular dynamics simulations have thus identified both sequence- and structure-specific differences in flexibility, long-range interactions, and rotameric form of key residues. Potential biological consequences of differential flexibility and long-range correlated motion are discussed.     

Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1

We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.     

Study on conformational homology of the HIV-1 gp120 protein V3 loop. Structural analysis of the HIV-RF and HIV-thailand viral strains

Based on NMR spectroscopy data, conformation of the HIV-RF gp120 protein V3 loop giving rise to the virus principal neutralizing determinant and also determinants of cell tropism and syncytium formation was calculated by computer modeling approaches. Elements of the HIV-RF V3 loop secondary structure and conformational states of its irregular stretches were determined. The calculated structure was compared with the conformation of the homologous stretch of the HIV-Thailand protein gp120 V3 loop, and structural elements preserved in the two viral strains were identified. Conservative elements of the HIV-1 V3 loop structure are considered to be promising targets for deriving chemically modified forms of this loop with the enhanced immunogenicity and cross-reactivity of neutralizing antibodies and also for creation of effective antiviral drugs on this base.     

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.     

Understanding the roles of amino acid residues in tertiary structure formation of chignolin by using molecular dynamics simulation

Chignolin is a 10-residue peptide (GYDPETGTWG) that forms a stable beta-hairpin structure in water. However, its design template, GPM12 (GYDDATKTFG), does not have a specific structure. To clarify which amino acids give it the ability to form the beta-hairpin structure, we calculated the folding free-energy landscapes of chignolin, GPM12, and their chimeric peptides using multicanonical molecular dynamics (MD) simulation. Cluster analysis of the conformational ensembles revealed that the native structure of chignolin was the lowest in terms of free energy while shallow local minima were widely distributed in the free energy landscape of GPM12, in agreement with experimental observations. Among the chimeric peptides, GPM12(D4P/K7G) stably formed the same beta-hairpin structure as that of chignolin in the MD simulation. This was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A comparison of the free-energy landscapes showed that the conformational distribution of the Asp3-Pro4 sequence was inherently biased in a way that is advantageous both to forming hydrogen bonds with another beta-strand and to initiating loop structure. In addition, Gly7 helps stabilize the loop structure by having a left-handed alpha-helical conformation. Such a conformation is necessary to complete the loop structure, although it is not preferred by other amino acids. Our results suggest that the consistency between the short-range interactions that determine the local geometries and the long-range interactions that determine the global structure is important for stable tertiary structure formation.     

Stabilization of the biologically active conformation of the principal neutralizing determinant of HIV-1(IIIB) containing a cis-proline surrogate: 1H NMR and molecular modeling study

The V3 loop is part of the gp120 glycoprotein, an extracellular protein located on the membrane of the human immunodeficiency virus (HIV-1). This loop is significantly important in many biological processes of the virus and contains the principal neutralizing determinant (PND). The PND is one of the most variable regions of the envelope, and this is probably related to the ability of the HIV virus to escape the immunologic defenses of the target host. Particular attention has been paid to the central part of the V3 loop which contains a highly conserved GPGR/GPGQ sequence and represents the binding site for antibodies. Many attempts have been made to design synthetic peptides as mimics of the V3 loop capable of eliciting immune response. However, this strategy suffers from the great conformational flexibility small peptides have in solution, which together with bioavailability represents the most important limitation to the usefulness of synthetic peptides as drugs and as synthetic immunogens. The use of conformationally constrained peptides can alleviate this problem. Early works using NMR studies have shown that a V3(IIIB) loop-derived peptide is conformationally heterogeneous when free in water. Upon complexation with 0.5beta, a monoclonal neutralizing antibody specific for the HIV-1(IIIB) strain, it adopts a beta-hairpin conformation with the central proline forming a type VIb beta-turn. In this study, we report the design and characterization of a conformationally restricted peptide with a sequence identical to that previously described, but with thiazolidine derivatives replacing the proline. The affinity of the 2,2-dimethylthiazolidine derivative for 0.5beta demonstrates that this moiety can successfully be used to mimic the proline in a cis conformation. This peptide not only displays a high propensity to adopt a beta-hairpin conformation but also retains the type VIb RGPG beta-turn similar to that found in the native complex. These compounds could help in elaborating more efficient immunogens for HIV-1 synthetic vaccine development.     

Hexapeptide analogue of somatostatin, Sandoz 201-456. Conformational study in dimethylsulfoxide by 1D and 2D n.m.r. methods

The conformational properties of the somatostatin analogue 201-456 (1) have been studied by high field n.m.r. in DMSO. This analogue is the base structure of nine derivates synthesized by Bauer et al. and shows a very low biological activity, although derived structures such as SMS 201-995 (2) are very potent. Our study has shown an important difference between the most stable conformation of the two compounds: although the beta turn type II' structure at the Phe3-Trp4-Lys5 level is present in both analogues, an important conformational change appears at the cystine bridge. In SMS 201-995 the beta turn/beta sheet conformation is stabilized by the additional amino-acids D-Phe1 and Thr8 (ol) through intramolecular H-bonds.     

Identification of the active conformation and the importance of length of the flexible loop 72-83 in regulating the conformational change of undecaprenyl pyrophosphate synthase

Increasing evidence has shown that intrinsic disorder of proteins plays a key role in their biological functions. In the case of undecaprenyl pyrophosphate synthase (UPPs), which catalyzes the chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate via eight consecutive condensation reactions with isopentenyl pyrophosphate, a highly flexible loop 72-83 was previously linked to protein conformational change required for catalysis [Chen, Y. H., Chen, A. P.-C., Chen, C. T., Wang, A. H.-J., and Liang, P. H., (2002) J. Biol. Chem. 277, 7369-7376]. The crystal structure and fluorescence studies suggested that the alpha3 helix connected to the loop moves toward the active site when the substrate is bound. To identify the active conformation and study the role of the loop for conformational change, the UPPs mutants with amino acids inserted into or deleted from the loop were examined. The inserted mutant with extra Ala residues fails to display the intrinsic fluorescence quenching upon FPP binding, and its crystal structure reveals only the open form. These phenomena appear to be different from the wild-type enzyme in which open and closed conformers were observed and suggest that the extended loop fails to pull the alpha3 helix and/or the extra amino acids in the loop cause steric hindrance on the alpha3 helix movement. The loop-shortening mutants with deletion of V82 and S83 or S72 also adopt an open conformation with the loop stretched, although they show decreased intrinsic fluorescence with FPP bound, similar to that seen in the wild-type enzyme. We conclude that the closed conformation is apparently the active conformation. Change of the length of the loop 72-83 impairs the ability of conformational change and causes remarkably lower activity of UPPs.     

The long and short flavodoxins: I. The role of the differentiating loop in apoflavodoxin structure and FMN binding

Flavodoxins are well known one-domain alpha/beta electron-transfer proteins that, according to the presence or absence of a approximately 20-residue loop splitting the fifth beta-strand of the central beta-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split beta-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184-47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.     

Model peptide studies of sequence regions in the elastomeric biomineralization protein, Lustrin A. I. The C-domain consensus-PG-, -NVNCT-motif

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomic force microscopy (AFM) pulling studies have demonstrated that the lustrin‐containing organic nacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force‐extension curve with hysteretic recovery. This force extension behavior is reminiscent of reversible unfolding and refolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays an important role in force‐induced protein unfolding and refolding, the question is, What secondary structure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKNVNCTSGE) representing the 12‐residue consensus sequence found near the N‐termini of the first eight cysteine‐rich domains (C‐domains) within the Lustrin A protein, we employed CD, NMR spectroscopy, and simulated annealing/minimization to determine the secondary structure preferences for this sequence. At pH 7.4, we find that the 12‐mer sequence adopts a loop conformation, consisting of a “bend” or “turn” involving residues G3–K4 and N7–C8–T9, with extended conformations arising at F1–G3; K4–V6; T9–S10–G11 in the sequence. Minor pH‐dependent conformational effects were noted for this peptide; however, there is no evidence for a salt‐bridge interaction between the K4 and E12 side chains. The presence of a loop conformation within the highly conserved —PG—, —NVNCT— sequence of C1–C8 domains may have important structural and mechanistic implications for the Lustrin A protein with regard to elastic behavior. © 2002 Wiley Periodicals, Inc. Biopolymers 63: 358–369, 2002     

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.     

Nuclear magnetic resonance structure of the Varkud satellite ribozyme stem-loop V RNA and magnesium-ion binding from chemical-shift mapping

An important step in the substrate recognition of the Neurospora Varkud Satellite (VS) ribozyme is the formation of a magnesium-dependent loop/loop interaction between the terminal loops of stem-loops I and V. We have studied the structure of stem-loop V by nuclear magnetic resonance spectroscopy and shown that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of stem-loop V fulfills some but not all of the structural characteristics found in canonical U-turn structures. This U-turn conformation exposes the Watson-Crick faces of the bases within stem-loop V (G697, A698, and C699) and makes them accessible for interaction with stem-loop I. Using chemical-shift mapping, we show that magnesium ions interact with the loop of the isolated stem-loop V and induce a conformational change that may be important for interaction with stem-loop I. This study expands our understanding of the role of U-turn motifs in RNA structure and function and provides insights into the mechanism of substrate recognition in the VS ribozyme.     

Early steps of the intramolecular signal transduction in rhodopsin explored by molecular dynamics simulations

We present molecular dynamics simulations of bovine rhodopsin in a membrane mimetic environment based on the recently refined X-ray structure of the pigment. The interactions between the protonated Schiff base and the protein moiety are explored both with the chromophore in the dark-adapted 11-cis and in the photoisomerized all-trans form. Comparison of simulations with Glu181 in different protonation states strongly suggests that this loop residue located close to the 11-cis bond bears a negative charge. Restrained molecular dynamics simulations also provide evidence that the protein tightly confines the absolute conformation of the retinal around the C12-C13 bond to a positive helicity. 11-cis to all-trans isomerization leads to an internally strained chromophore, which relaxes after a few nanoseconds by a switching of the ionone ring to an essentially planar all-trans conformation. This structural transition of the retinal induces in turn significant conformational changes of the protein backbone, especially in helix VI. Our results suggest a possible molecular mechanism for the early steps of intramolecular signal transduction in a prototypical G-protein-coupled receptor.     

Conformational preferences of two peptides DYASL and DYA from haemagglutinin of influenza virus and their possible role in the initiation of protein folding

The conformational preferences of two peptides DYASL and DYA from haemagglutinin of influenza virus were studied using PCILO programme. This was done to understand the possible role of DYAS in the initiation of protein folding and to understand the contribution of the fourth residue serine in the formation of turn. Our results indicate that this sequence shows an inherent preference for turn conformation, with a stabilizing Asx turn. DYA with NH group in the C-terminal protection models a type I beta turn more closelythan DYAS, because serine has a weak potential for turn conformation.     

Conformational and sequence signatures in beta helix proteins

beta helix proteins are characterized by a repetitive fold, in which the repeating unit is a beta-helical coil formed by three strand segments linked by three loop segments. Using a data set of left- and right-handed beta helix proteins, we have examined conformational features at equivalent positions in successive coils. This has provided insights into the conformational rules that the proteins employ to fold into beta helices. Left-handed beta helices attain their equilateral prism fold by incorporating "corners" with the conformational sequence P(II)-P(II)-alpha(L)-P(II), which imposes sequence restrictions, resulting in the first and third P(II) residues often being G and a small, uncharged residue (V, A, S, T, C), respectively. Right-handed beta helices feature mid-sized loops (4, 5, or 6 residues) of conserved conformation, but not of conserved sequence; they also display an alpha-helical residue at the C-terminal end of L2 loops. Backbone conformational parameters (phi,psi) that permit the formation of continuous, loopless beta helices (Perutz nanotubes) have also been investigated.     

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.     

Molecular basis for the Cu2+ binding-induced destabilization of beta2-microglobulin revealed by molecular dynamics simulation

According to experimental data, binding of the Cu(2+) ions destabilizes the native state of beta2-microglobulin (beta2m). The partial unfolding of the protein was generally considered an early step toward fibril formation in dialysis-related amyloidosis. Recent NMR studies have suggested that the destabilization of the protein might be achieved through increased flexibility upon Cu(2+) binding. However, the molecular mechanism of destabilization due to Cu(2+), its role in amyloid formation, and the relative contributions of different potential copper-binding sites remain unclear. To elucidate the effect of ion ligation at atomic detail, a series of molecular dynamics simulations were carried out on apo- and Cu(2+)-beta2m systems in explicit aqueous solutions, with varying numbers of bound ions. Simulations at elevated temperatures (360 K) provide detailed pictures for the process of Cu(2+)-binding-induced destabilization of the native structure at the nanosecond timescale, which are in agreement with experiments. Conformational transitions toward partially unfolded states were observed in protein solutions containing bound copper ions at His-31 and His-51, which is marked by an increase in the protein vibrational entropy, with TDeltaS(vibr) ranging from 30 to 69 kcal/mol. The binding of Cu(2+) perturbs the secondary structure and the hydrogen bonding pattern disrupts the native hydrophobic contacts in the neighboring segments, which include the beta-strand D2 and part of the beta-strand E, B, and C and results in greater exposure of the D-E loop and the B-C loop to the water environment. Analysis of the MD trajectories suggests that the changes in the hydrophobic environment near the copper-binding sites lower the barrier of conformational transition and stabilize the more disordered conformation. The results also indicate that the binding of Cu(2+) at His-13 has little effect on the conformational stability, whereas the copper-binding site His-31, and to a lesser extent His-51, are primarily responsible for the observed changes in the protein conformation and dynamics.     

The Influence of N-Linked Glycans on the Molecular Dynamics of the HIV-1 gp120 V3 Loop

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.     

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease.