The investigation of the secondary structure propensities and free-energy landscapes of peptide ligands by replica exchange molecular dynamics simulations

The conformational states of two peptide sequences that bind to staphylococcal enterotoxin B are sampled by replica exchange molecular dynamic (REMD) simulations in explicit water. REMD simulations were treated with 52 replicas in the range of 280–501 K for both peptides. The conformational ensembles of both peptides are dominated by random coil, bend and turn structures with a small amount of helical structures for each temperature. In addition, while an insignificant presence of β-bridge structures were observed for both peptides, the β-sheet structure was observed only for peptide 3. The results obtained from simulations at 300 K are consistent with the experimental results obtained from circular dichroism spectroscopy. From the analysis of REMD results, we also calculated hydrophobic and hydrophilic solvent accessible surface areas for both peptides, and it was observed that the hydrophobic segments of the peptides tend to form bend or turn structures. Moreover, the free-energy landscapes of both peptides were obtained by principal component analysis to understand how the secondary structural properties change according to their complex space. From the free-energy analysis, we have found several minima for both peptides at decreased temperature. For these obvious minima of both peptides, it was observed that the random coil, bend and turn structures are still dominant and the helix, β-bridge or β-sheet structures can appear or disappear with respect to minima. On the other hand, when we compare the results of REMD with conventional MD simulations for these peptides, the configurations of peptide 3 might be trapped in energy minima during the conventional MD simulations. Hence, it can be said that the REMD simulations have provided a sufficiently high sampling efficiency.     

Helix-coil transition of alanine peptides in water: force field dependence on the folded and unfolded structures

The force fields used in classical modeling studies are semiempirical in nature and rely on their validation by comparison of simulations with experimental data. The all-atom replica-exchange molecular dynamics (REMD) methodology allows us to calculate the thermodynamics of folding/unfolding of peptides and small proteins, and provides a way of evaluating the reliability of force fields. We apply the REMD to obtain equilibrium folding/unfolding thermodynamics of a 21-residue peptide containing only alanine residues in explicit aqueous solution. The thermodynamics of this peptide is modeled with both the OPLS/AA/L and the A94/MOD force fields. We find that the helical content and the values for the helix propagation and nucleation parameters for this alanine peptide are consistent with measurements on similar peptides and with calculations using the modified AMBER force field (A94/MOD). The nature of conformations, both folded and unfolded, that contributes to the helix-coil transition profile, however, is quite different between these two force fields.     

Structural dynamics of the actin-myosin interface by site-directed spectroscopy

We have used site-directed spin and fluorescence labeling to test molecular models of the actin-myosin interface. Force is generated when the actin-myosin complex undergoes a transition from a disordered weak-binding state to an ordered strong-binding state. Actomyosin interface models, in which residues are classified as contributing to either weak or strong binding, have been derived by fitting individual crystallographic structures of actin and myosin into actomyosin cryo-EM maps. Our goal is to test these models using site-directed spectroscopic probes on actin and myosin. Starting with Cys-lite constructs of both yeast actin (ActC) and the Dictyostelium myosin II motor domain (S1dC), site-directed labeling (SDL) mutants were generated by mutating residues to Cys in the proposed weak and strong-binding interfaces. This report focuses on the effects of forming the strong-binding complex on four SDL mutants, two located in the proposed weak-binding interface (ActC5 and S1dC619) and two located in the proposed strong-binding interface (ActC345 and S1dC401). Neither the mutations nor labeling prevented strong actomyosin binding or actin-activation of myosin ATPase. Formation of the strong-binding complex resulted in decreased spin and fluorescence probe mobility at all sites, but both myosin-bound probes showed remarkably high mobility even after complex formation. Complex formation decreased solvent accessibility for both actin-bound probes, but increased it for the myosin-bound probes. These results are not consistent with a simple model in which there are discrete weak and strong interfaces, with only the strong interface forming under strong-binding conditions, nor are they consistent with a model in which surface residues become rigid and inaccessible upon complex formation. We conclude that all four of these residues are involved in the strong actin-myosin interface, but this interface is remarkably dynamic, especially on the surface of myosin.     

Structures of designed armadillo repeat proteins binding to peptides fused to globular domains

Designed armadillo repeat proteins (dArmRP) are α‐helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term “peptide” not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N‐terminus of Green Fluorescent Protein or to the C‐terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein–peptide interactions are characterized, and should always be correlated with measurements in solution.     

Quantification of protein-protein interactions with chemical cross-linking and mass spectrometry

Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein-protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein-protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin-melittin and calmodulin-mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin-melittin and three interpeptide cross-links for calmodulin-mastoparan. Using cross-linking sites of calmodulin-mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. We also demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.     

The Alzheimer's peptides Abeta40 and 42 adopt distinct conformations in water: a combined MD / NMR study

The role of peptides Abeta40 and Abeta42 in the early pathogenesis of Alzheimer's disease (AD) is frequently emphasized in the literature. It is known that Abeta42 is more prone to aggregation than Abeta40, even though they differ in only two (IA) amino acid residues at the C-terminal end. A direct comparison of the ensembles of conformations adopted by the monomers in solution has been limited by the inherent flexibility of the unfolded peptides. Here, we characterize the conformations of Abeta40 and Abeta42 in water by using a combination of molecular dynamics (MD) and measured scalar (3)J(HNHalpha) data from NMR experiments. We perform replica exchange MD (REMD) simulations and find that classical forcefields reproduce the NMR data quantitatively when the sampling is extended to the microseconds time-scale. Using the quantitative agreement of the NMR data as a validation of the model, we proceed to compare the conformational ensembles of the Abeta40 and Abeta42 peptide monomers. Our analysis confirms the existence of structured regions within the otherwise flexible Abeta peptides. We find that the C terminus of Abeta42 is more structured than that of Abeta40. The formation of a beta-hairpin in the sequence (31)IIGLMVGGVVIA involving short strands at residues 31-34 and 38-41 (in bold) reduces the C-terminal flexibility of the Abeta42 peptide and may be responsible for the higher propensity of this peptide to form amyloids.     

Tryptophan rich peptides: influence of indole rings on backbone conformation

Synthetic peptides with defined secondary structure scaffolds, namely hairpins and helices, containing tryptophan residues, have been investigated in this study to probe the influence of a large number of aromatic amino acids on backbone conformations. Solution NMR investigations of Boc-W-L-W-(D)P-G-W-L-W-OMe (peptide 1), designed to form a well-folded hairpin, clearly indicates the influence of flanking aromatic residues at the (D)Pro-Gly region on both turn nucleation and strand propagation. Indole-pyrrolidine interactions in this peptide lead to the formation of the less-frequent type I' turn at the (D)Pro-Gly segment and frayed strand regions, with the strand residues adopting local helical conformations. An analog of peptide 1 with an Aib-Gly turn-nucleated hairpin (Boc-W-L-W-U-G-W-L-W-OMe (peptide 2)) shows a preference for helical structures in solution, in both chloroform and methanol. Peptides with either one (Boc-W-L-W-U-W-L-W-OMe (peptide 3)) or two (Boc-U-W-L-W-U-W-L-W-OMe (peptide 4)) helix-nucleating Aib residues give rise to the well-folded helical conformations in the chloroform solution. The results are indicative of a preference for helical folding in peptides containing a large number of Trp residues. Investigation of a tetrapeptide analog of peptide 2, Boc-W-U-G-W-OMe (peptide 5), in solution and in the crystal state (by X-ray diffraction), also indicates a preference for a helical fold. Additionally, peptide 5 is stabilized in crystals by both aromatic interactions and an array of weak interactions. Examination of Trp-rich sequences in protein structures, however, reveals no secondary structure preference, suggesting that other stabilizing interactions in a well-folded protein may offset the influence of indole rings on backbone conformations.     

Plasticity in protein-peptide recognition: crystal structures of two different peptides bound to concanavalin A

The structures of concanavalin A (ConA) in complex with two carbohydrate-mimicking peptides, 10-mer (MYWYPYASGS) and 15-mer (RVWYPYGSYLTASGS) have been determined at 2.75 A resolution. In both crystal structures four independent peptide molecules bind to each of the crystallographically independent subunits of ConA tetramer. The peptides exhibit small but significant variability in conformations and interactions while binding to ConA. The crystal structure of another similar peptide, 12-mer (DVFYPYPYASGS), in complex with ConA has been determined (Jain, D., K. J. Kaur, B. Sundaravadivel, and D. M. Salunke. 2000. Structural and functional consequences of peptide-carbohydrate mimicry. J. Biol. Chem. 275:16098-16102). Comparison of the three complexes shows that the peptides bind to ConA at a common binding site, using different contacting residues and interactions depending on their sequence and the local environment at the binding site. The binding is also optimized by corresponding plasticity of the peptide binding site on ConA. The diversity in conformation and interactions observed here are in agreement with the structural leeway concerning plasticity of specific molecular recognition in biological processes. The adaptability of peptide-ConA interactions may also be correlated with the carbohydrate-mimicking property of these peptides.     

Amino acid requirement for the high affinity binding of a selected arginine-rich peptide with the HIV Rev-response element RNA.

The arginine-rich motif is a class of short arginine-rich peptides that bind to specific RNA structures that has been found to be a versatile framework for the design and selection of RNA-binding peptides. We previously identified novel peptides that bind to the Rev-response element (RRE) RNA of the HIV from an arginine-rich polypeptide library (ARPL) consisting of a polyarginine (15 mer) randomized at the N-terminal 10 positions. The selected peptides bound more strongly to the RRE than the natural binding partner, Rev, and contained glutamine residues that were assumed to be important for recognition of the G-A base pair. In addition, the peptides were predicted to bind to the RRE in an alpha-helical conformation. In this study, in order to understand the mechanism of the interaction between the RRE and the putative alpha-helical glutamine-containing peptides, the amino acid requirements for high affinity binding were analyzed by a combinatorial approach using a bacterial system for detecting RNA-peptide interactions. A consensus peptide, the DLA peptide, was elucidated, which consists of a single glutamine residue within a polyarginine context with the glutamine residue flanked at specific positions by three nonarginine residues, two of which appear to be important for alpha-helix stabilization. In addition, the DLA peptide was found to bind extremely tightly to the RRE with an affinity 50-fold higher than that of the Rev peptide as determined by a gel shift assay. A working model for the interaction of the DLA peptide to the RRE is proposed, which should aid in the development of peptide-based drugs that inhibit HIV replication, as well as in our understanding of polypeptide-RNA interactions. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Formation of partially ordered oligomers of amyloidogenic hexapeptide (NFGAIL) in aqueous solution observed in molecular dynamics simulations

A combined total of more than 600.0 ns molecular dynamics simulations with explicit solvent have been carried on systems containing either four peptides or a single peptide to investigate the early-stage aggregation process of an amyloidogenic hexapeptide, NFGAIL (residues 22-27 of the human islet amyloid polypeptide). Direct observation of the aggregation process was made possible by placing four peptides in a box of water with an effective concentration of 158 mg/ml to enhance the rate of aggregation. Partially ordered oligomers containing multistrand beta-sheets were observed which could be the precursory structures leading to the amyloid-forming embryonic nuclei. Comparative simulations on a single peptide suggested that the combined effect of higher peptide concentration and periodic boundary condition promoted compact monomers and the short-range interpeptide interactions favored the beta-extended conformation. Of particular interest was the persistent fluctuation of the size of the aggregates throughout the simulations, suggesting that dissociation of peptides from the disordered aggregates was an obligatory step toward the formation of ordered oligomers. Although 95% of peptides formed oligomers and 44% were in beta-extended conformations, only 16% of peptides formed multistrand beta-sheets. The disordered aggregates were mainly stabilized by hydrophobic interactions while cross-strand main-chain hydrogen bonds manifested the ordered oligomers. The transition to the beta-extended conformation was mildly cooperative due to short-range interactions between beta-extended peptides. Taken together, we propose that the role of hydrophobic interaction in the early stage of aggregation is to promote disordered aggregates and disfavor the formation of ordered nuclei and dissociation of the disordered oligomers could be the rate-limiting step at the initiation stage.     

Coarse-grained simulation studies of peptide-induced pore formation

We investigate the interactions between lipid bilayers and amphiphilic peptides using a solvent-free coarse-grained simulation technique. In our model, each lipid is represented by one hydrophilic and three hydrophobic beads. The amphiphilic peptide is modeled as a hydrophobic-hydrophilic cylinder with hydrophilic caps. We find that with increasing peptide-lipid attraction the preferred state of the peptide changes from desorbed, to adsorbed, to inserted. A single peptide with weak attraction binds on the bilayer surface, while one with strong attraction spontaneously inserts into the bilayer. We show how several peptides, which individually bind only to the bilayer surface, cooperatively insert. Furthermore, hydrophilic strips along the peptide cylinder induce the formation of multipeptide pores, whose size and morphology depend on the peptides’ overall hydrophilicity, the distribution of hydrophilic residues, and the peptide-peptide interactions. Strongly hydrophilic peptides insert less readily, but prove to be more destructive to bilayer integrity.     

A novel knowledge-based approach to design inorganic-binding peptides

MOTIVATION: The discovery of solid-binding peptide sequences is accelerating along with their practical applications in biotechnology and materials sciences. A better understanding of the relationships between the peptide sequences and their binding affinities or specificities will enable further design of novel peptides with selected properties of interest both in engineering and medicine. RESULTS: A bioinformatics approach was developed to classify peptides selected by in vivo techniques according to their inorganic solid-binding properties. Our approach performs all-against-all comparisons of experimentally selected peptides with short amino acid sequences that were categorized for their binding affinity and scores the alignments using sequence similarity scoring matrices. We generated novel scoring matrices that optimize the similarities within the strong-binding peptide sequences and the differences between the strong- and weak-binding peptide sequences. Using the scoring matrices thus generated, a given peptide is classified based on the sequence similarity to a set of experimentally selected peptides. We demonstrate the new approach by classifying experimentally characterized quartz-binding peptides and computationally designing new sequences with specific affinities. Experimental verifications of binding of these computationally designed peptides confirm our predictions with high accuracy. We further show that our approach is a general one and can be used to design new sequences that bind to a given inorganic solid with predictable and enhanced affinity.     

A potent new class of reductively activated peptide gene delivery agents

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.     

Structure and energy landscape of a photoswitchable peptide: a replica exchange molecular dynamics study

A replica exchange molecular dynamics (REMD) simulation of a bicyclic azobenzene peptide in explicit dimethyl sulfoxide solution is presented in order to characterize the conformational structures and energy landscape of a photoswitchable peptide. It is shown that an enhanced-sampling technique such as the REMD method is essential to obtain a converged conformational sampling of the peptide at room temperature. This is because conventional MD simulations of less than approximately 100-ns length are either trapped in local minima (at 295 K) or-if run at high temperature-do not resemble the room-temperature REMD results. Calculating various nuclear Overhauser effects (NOEs) and (3)J-couplings, a good overall agreement between the REMD simulations and the NMR experiments of Renner et al. (Biopolymers 2000;54:501-514) is found. In particular, the REMD study confirms the general picture drawn by Renner et al. that the trans-isomer of the azobenzene peptide exhibits a well-defined structure, while the cis-isomer is a conformational heterogeneous system; that is, the trans-isomer occurs in 2 well-defined conformers, while the cis-isomer represents an energetically frustrated system that leads to an ensemble of conformational structures. Employing a principal component analysis of the REMD data, the free energy landscape of the systems is studied at various temperatures. The implications for the folding and unfolding pathways of the system are discussed.     

Molecular recognition of p53 and MDM2 by USP7/HAUSP

The ubiquitin-specific protease, USP7, has key roles in the p53 pathway whereby it stabilizes both p53 and MDM2. We show that the N-terminal domain of USP7 binds two closely spaced 4-residue sites in both p53 and MDM2, falling between p53 residues 359-367 and MDM2 residues 147-159. Cocrystal structures with USP7 were determined for both p53 peptides and for one MDM2 peptide. These peptides bind the same surface of USP7 as Epstein-Barr nuclear antigen-1, explaining the competitive nature of the interactions. The structures and mutagenesis data indicate a preference for a P/AXXS motif in peptides that bind USP7. Contacts made by serine are identical and crucial for all peptides, and Trp165 in the peptide-binding pocket of USP7 is also crucial. These results help to elucidate the mechanism of substrate recognition by USP7 and the regulation of the p53 pathway.     

Mechanical unbinding of abeta peptides from amyloid fibrils

Using the experimental structures of Abeta amyloid fibrils and all-atom molecular dynamics, we study the force-induced unbinding of Abeta peptides from the fibril. We show that the mechanical dissociation of Abeta peptides is highly anisotropic and proceeds via different pathways when force is applied in parallel or perpendicular direction with respect to the fibril axis. The threshold forces associated with lateral unbinding of Abeta peptides exceed those observed during the mechanical dissociation along the fibril axis. In addition, Abeta fibrils are found to be brittle in the lateral direction of unbinding and soft along the fibril axis. Lateral mechanical unbinding and the unbinding along the fibril axis load different types of fibril interactions. Lateral unbinding is primarily determined by the cooperative rupture of fibril backbone hydrogen bonds. The unbinding along the fibril axis largely depends on the interpeptide Lys-Asp electrostatic contacts and the hydrophobic interactions formed by the Abeta C terminal. Due to universality of the amyloid beta structure, the anisotropic mechanical dissociation observed for Abeta fibrils is likely to be applicable to other amyloid assemblies. The estimates of equilibrium forces required to dissociate Abeta peptide from the amyloid fibril suggest that these supramolecular structures are mechanically stronger than most protein domains.     

Mapping Conformational Ensembles of Aβ Oligomers in Molecular Dynamics Simulations

Although the oligomers formed by Aβ peptides appear to be the primary cytotoxic species in Alzheimer's disease, detailed information about their structures appears to be lacking. In this article, we use exhaustive replica exchange molecular dynamics and an implicit solvent united-atom model to study the structural properties of Aβ monomers, dimers, and tetramers. Our analysis suggests that the conformational ensembles of Aβ dimers and tetramers are very similar, but sharply distinct from those sampled by the monomers. The key conformational difference between monomers and oligomers is the formation of β-structure in the oligomers occurring together with the loss of intrapeptide interactions and helix structure. Our simulations indicate that, independent of oligomer order, the Aβ aggregation interface is largely confined to the sequence region 10-23, which forms the bulk of interpeptide interactions. We show that the fractions of β structure computed in our simulations and measured experimentally are in good agreement.     

Interpeptide interactions induce helix to strand structural transition in Aβ peptides

Replica exchange molecular dynamics and all‐atom implicit solvent model are used to compute the structural propensities in Aβ monomers, dimers, and Aβ peptides bound to the edge of amyloid fibril. These systems represent, on an approximate level, different stages in Aβ aggregation. Aβ monomers are shown to form helical structure in the N‐terminal (residues 13 to 21). Interpeptide interactions in Aβ dimers and, especially, in the peptides bound to the fibril induce a dramatic shift in the secondary structure, from helical states toward β‐strand conformations. The sequence region 10–23 in Aβ peptide is found to form most of interpeptide interactions upon aggregation. Simulation results are tested by comparing the chemical shifts in Aβ monomers computed from simulations and obtained experimentally. Possible implications of our simulations for designing aggregation‐resistant variants of Aβ are discussed. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Secondary structures and intramolecular interactions in fragments of the B-loops of naturally occurring analogs of epidermal growth factor

Structures of naturally occurring analogs of the B-loop fragment of human epidermal growth factor-like (hEGF-like) polypeptides were examined by molecular dynamics simulation in order to predict their secondary structures, to find structural similarity and to detect any weakly polar aromatic-aromatic (pi-pi) or amide-aromatic (N-pi) interactions which stabilize the structures. NPT molecular dynamics simulations (1 ns) were performed by the GRO-MACS package with SPC/E water using a weak temperature and pressure coupling method. During the sampling time, the structures of all peptides showed a characteristic secondary structure with a turn and bend at residues 4-7, and a beta-sheet, beta-bridge and random coil at the N- and C-terminal regions. Though the peptide chains were flexible, the stabilization effect of the N-pi interactions was indicated in some cases by the angles and distances between the centroids of aromatic planes of the side-chains and the H-atom of peptide bonds and the planes of the aromatic side-chains, respectively. Pi-pi interactions occurred less frequently because of the flexibility of the short peptide chain.     

Free energy landscapes for amyloidogenic tetrapeptides dimerization

The oligomerization of four peptide sequences, KFFE, KVVE, KLLE, and KAAE is studied using replica-exchange molecular dynamics simulations with an atomically detailed peptide model. Previous experimental studies reported that of these four peptides, only those containing phenylalanine and valine residues form fibrils. We show that the fibrillogenic propensities of these peptides can be rationalized in terms of the equilibrium thermodynamics of their early oligomers. Thermodynamic stability of dimers, as measured by the temperature of monomer association, is seen to be higher for those peptides that are able to form fibrils. Although the relative high and low stabilities of the KFFE and KAAE dimers arise from their respective high and low interpeptide interaction energies, the higher stability of the KVVE dimer over the KLLE system results from the smaller loss of configurational entropy accompanying the dimerization of KVVE. Free energy landscapes for dimerization are found to be strongly sequence-dependent, with a high free energy barrier separating the monomeric and dimeric states for KVVE, KLLE, and KAAE sequences. In contrast, the most fibrillogenic peptide, KFFE, displayed downhill assembly, indicating enhanced kinetic accessibility of its dimeric states. The dimeric phase for all peptide sequences is found to be heterogeneous, containing both antiparallel beta-sheet structures that can grow into full fibrils as well as disordered dimers acting as on- or off-pathway intermediates for fibrillation.