The use of in vitro peptide binding profiles and in silico ligand-receptor interaction profiles to describe ligand-induced conformations of the retinoid X receptor alpha ligand-binding domain

It is hypothesized that different ligand-induced conformational changes can explain the different interactions of nuclear receptors with regulatory proteins, resulting in specific biological activities. Understanding the mechanism of how ligands regulate cofactor interaction facilitates drug design. To investigate these ligand-induced conformational changes at the surface of proteins, we performed a time-resolved fluorescence resonance energy transfer assay with 52 different cofactor peptides measuring the ligand-induced cofactor recruitment to the retinoid X receptor-alpha (RXRalpha) in the presence of 11 compounds. Simultaneously we analyzed the binding modes of these compounds by molecular docking. An automated method converted the complex three-dimensional data of ligand-protein interactions into two-dimensional fingerprints, the so-called ligand-receptor interaction profiles. For a subset of compounds the conformational changes at the surface, as measured by peptide recruitment, correlate well with the calculated binding modes, suggesting that clustering of ligand-receptor interaction profiles is a very useful tool to discriminate compounds that may induce different conformations and possibly different effects in a cellular environment. In addition, we successfully combined ligand-receptor interaction profiles and peptide recruitment data to reveal structural elements that are possibly involved in the ligand-induced conformations. Interestingly, we could predict a possible binding mode of LG100754, a homodimer antagonist that showed no effect on peptide recruitment. Finally, the extensive analysis of the peptide recruitment profiles provided novel insight in the potential cellular effect of the compound; for the first time, we showed that in addition to the induction of coactivator peptide binding, all well-known RXRalpha agonists also induce binding of corepressor peptides to RXRalpha.     

Differential association of HLA-B*2705 and B*2709 to ankylosing spondylitis correlates with limited peptide subsets but not with altered cell surface stability

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to beta2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype.     

Structural prediction of peptides binding to MHC class I molecules

Peptide binding to class I major histocompatibility complex (MHCI) molecules is a key step in the immune response and the structural details of this interaction are of importance in the design of peptide vaccines. Algorithms based on primary sequence have had success in predicting potential antigenic peptides for MHCI, but such algorithms have limited accuracy and provide no structural information. Here, we present an algorithm, PePSSI (peptide-MHC prediction of structure through solvated interfaces), for the prediction of peptide structure when bound to the MHCI molecule, HLA-A2. The algorithm combines sampling of peptide backbone conformations and flexible movement of MHC side chains and is unique among other prediction algorithms in its incorporation of explicit water molecules at the peptide-MHC interface. In an initial test of the algorithm, PePSSI was used to predict the conformation of eight peptides bound to HLA-A2, for which X-ray data are available. Comparison of the predicted and X-ray conformations of these peptides gave RMSD values between 1.301 and 2.475 A. Binding conformations of 266 peptides with known binding affinities for HLA-A2 were then predicted using PePSSI. Structural analyses of these peptide-HLA-A2 conformations showed that peptide binding affinity is positively correlated with the number of peptide-MHC contacts and negatively correlated with the number of interfacial water molecules. These results are consistent with the relatively hydrophobic binding nature of the HLA-A2 peptide binding interface. In summary, PePSSI is capable of rapid and accurate prediction of peptide-MHC binding conformations, which may in turn allow estimation of MHCI-peptide binding affinity.     

The Relationship Between Structure and Activity Among Opioid Peptides

Summary Since the discovery and isolation of the endogenous opioid peptides Leu- and Met-enkephalin, structural studies have been focused on deducing the bioactive conformation of the peptide ligands. Theoretically, linear peptides can have many different backbone conformations, yet early, X-ray studies on enkephalin and its analogues showed only two different backbone conformations: extended and single β-bend. More recent reports include a third conformation for Leu-enkephalin and constrained opioid peptides from two ‘new’ classes (i.e. cyclic and ‘allaromatic’ peptides). In this report the relationship between solid-state X-ray structure and opioid peptide activity is examined. The N-terminal amine nitrogen and the two aromatic rings have previously been identified as structural features important to the biological activity of opioid peptides. From X-ray studies we find that the distances between the centroids of the aromatic rings, and between the N-terminal amine nitrogen and the centroid of the phenylalanine ring, vary over a large range. There is a discernible relationship, however, between the separation of the two rings and their orientation that correlates with activity.     

Structure and orientation of peptide inhibitors bound to beta-amyloid fibrils

Polymerization of the soluble beta-amyloid peptide into highly ordered fibrils is hypothesized to be a causative event in the development of Alzheimer's disease. Understanding the interactions of Abeta with inhibitors on an atomic level is fundamental for the development of diagnostics and therapeutic approaches, and can provide, in addition, important indirect information of the amyloid fibril structure. We have shown recently that trRDCs can be measured in solution state NMR for peptide ligands binding weakly to amyloid fibrils. We present here the structures for two inhibitor peptides, LPFFD and DPFFL, and their structural models bound to fibrillar Abeta(14-23) and Abeta(1-40) based on transferred nuclear Overhauser effect (trNOE) and transferred residual dipolar coupling (trRDC) data. In a first step, the inhibitor peptide structure is calculated on the basis of trNOE data; the trRDC data are then validated on the basis of the trNOE-derived structure using the program PALES. The orientation of the peptide inhibitors with respect to Abeta fibrils is obtained from trRDC data, assuming that Abeta fibrils orient such that the fibril axis is aligned in parallel with the magnetic field. The trRDC-derived alignment tensor of the peptide ligand is then used as a restraint for molecular dynamics docking studies. We find that the structure with the lowest rmsd value is in agreement with a model in which the inhibitor peptide binds to the long side of an amyloid fibril. Especially, we detect interactions involving the hydrophobic core, residues K16 and E22/D23 of the Abeta sequence. Structural differences are observed for binding of the inhibitor peptide to Abeta14-23 and Abeta1-40 fibrils, respectively, indicating different fibril structure. We expect this approach to be useful in the rational design of amyloid ligands with improved binding characteristics.     

Expression and Purification of Isotopically Enriched MHC Binding Immunogenic Peptides for NMR Studies

Peptides are important naturally occurring ligands of MHC molecules. X-ray crystallographic studies have enabled extensive characterization of such peptide ligands. Yet structural and dynamic changes of these peptides in the MHC bound state are not well understood. These conformational transitions are key to understanding the function of MHC molecules and for the development of peptide-based therapeutics. Employing NMR for such studies can fill this gap but it requires the availability of peptides labeled with NMR-active nuclei. Here we report production of nine-mer MHC-binding peptides for use in high resolution NMR studies. The method utilizes a fusion protein approach of attaching the peptide to an easily expressed bacterial protein. The fusion protein construct design allows for rapid purification of the fusion protein and avoids chemical modification of the peptide as a result of the cleavage reaction. The methods developed here allow for rapid cloning of additional MHC binding peptides without significant molecular biology effort. 8?C10 mg of mature freeze dried peptides can be obtained from 1 liter of minimal media, sufficient for NMR experimentation. Six uniformly ¹⁵N-labeled peptides have been successfully expressed in bacteria and NMR spectra with the expected number of well-resolved signals were recorded. The results obtained here will make peptide-MHC complexes amenable to structural analysis which has not been possible previously.     

All-Atom Monte Carlo Approach to Protein-Peptide Binding

We develop a procedure for exploring the free energy landscape of protein-peptide binding at atomic detail and apply it to PDZ domain-peptide interactions. The procedure involves soft constraints on receptor proteins providing limited chain flexibility, including backbone motions. Peptide chains are left fully flexible and kept in spatial proximity of the protein through periodic boundary conditions. By extensive Monte Carlo simulations, full representative conformational ensembles at temperatures where bound and unbound states coexist are obtained. To make this approach computationally feasible, we develop an effective all-atom energy function centering on hydrophobicity, hydrogen bonding, and electrostatic interactions. Our initial focus is a set of 11 PDZ domain-peptide pairs with experimentally determined complex structures. Minimum-energy conformations are found to be highly similar to the respective native structures in eight of the cases (all-atom peptide RMSDs < 6 Å). Having achieved that, we turn to a more complete characterization of the bound peptide state through a clustering scheme applied on the full ensembles of peptide structures. We find a significant diversity among bound peptide conformations for several PDZ domains, in particular involving the N terminal side of the peptide chains. Our computational model is then tested further on a set of nine PDZ domain-peptide pairs where the peptides are not originally present in the experimentally determined structures. We find a similar success rate in terms of the nativeness of minimum-energy conformations. Finally, we investigate the ability of our approach to capture variations in binding affinities for different peptide sequences. This is done in particular for a set of related sequences binding to the third PDZ domain of PSD-95 with encouraging results.     

Conformational Determinants of Phosphotyrosine Peptides Complexed with the Src SH2 Domain

The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA) to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the “two-pronged plug two-hole socket” model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an α-helix. In contrast, a β-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.     

Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors

Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions.     

Simulated dipeptide recognition by vancomycin

The antimicrobial activity of vancomycin and related glycopeptide antibiotics is due to stereospecific recognition of polypeptide components in bacterial cell walls. To better understand how these antibiotics recognize polypeptide determinants, we have developed dynamic models of the complexes formed by the vancomycin aglycon and two different dipeptide ligands, Ac-D-ala-D-ala and Ac-D-ala-gly. Molecular dynamics simulations of the two complexes, initially conditioned with distance constraints derived from two-dimensional nuclear magnetic resonance (NMR) studies, are conformationally stable and propagate in a manner consistent with the NMR-derived constraints after the constraints are removed. Free energy calculations accurately predict the relative binding affinity of these two complexes and help validate the simulation models for detailed structural analysis. Although the two ligands adopt similar conformations when bound to the antibiotic, there are clear differences in the configuration of intermolecular hydrogen bonds, the overall shape of the antibiotic, and other structural features of the two complexes. This analysis illustrates how complex structural and dynamic factors interrelate and contribute to differences in binding affinity. © 1997 John Wiley & Sons, Ltd.     

Conformational studies of antimetastatic laminin-1 derived peptides in different solvent systems, using solution NMR spectroscopy.

Due to its critical role in cancer progression, interactions between laminin-1 and the 67 kDa Laminin-Binding Protein (the 67 kDa LBP) have been the focus of a number of structural and biological studies. As laminin-1 is such a large and complex molecule, research interests have turned to the investigation of bioactive peptides derived from binding domains of laminin-1. Two peptides of interest, CDPGYIGSR (peptide 11) and YIGSR, both derived from the beta1 chain of laminin-1, have been shown to block invasion of basement membranes by tumor cells. Substituting the C-terminal arginine to lysine, a conservative substitution, results in a loss of peptide antimetastatic activity. This difference in bioactivity has been attributed, based on numerous modeling studies of free peptide conformations, to structural differences between YIGSR and YIGSK. Yet the nature of the 'active' free peptide backbone conformation has been a matter of debate and controversy. In order to test the validity of the structural modeling claims, we have undertaken detailed conformational studies of the two laminin-1 derived peptides YIGSR and CDPGYIGSR along with the biologically inactive YIGSK analog by two-dimensional solution 1H NMR spectroscopy in three different solvent systems. Herein we report that although both the active (YIGSR, CDPGYIGSR) and the inactive (YIGSK) peptides can adopt several closely related conformations in solution, the two peptides share similar conformational preferences, and there are no significant structural differences between the active and inactive peptides, contrary to previously reported modeling data. We conclude that the basis of the peptide biological activity, in contrast to published models, cannot be attributed to well-defined structural preferences of the free peptides. We infer that the difference in bioactivity observed between YIGSR and YIGSK originates primarily from the chemical nature of the arginine versus lysine sidechain substitution, rather than being due to a structural change in the free peptide conformations.     

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.     

Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10⁵ M^?1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright © 2009 John Wiley & Sons, Ltd.     

High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major "bottleneck" of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred "en masse" from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide-BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide-BAP protein can find direct application as a tracer reagent.     

Rhodopsin-transducin interface: studies with conformationally constrained peptides.

To probe the interaction between transducin (G(t)) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the C-terminal 11 residues of G(t)alpha derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G(t)alpha complex showed strong correlation to the experimental data.     

Structural resiliency of an EGF-like subdomain bound to its target protein, thrombin.

The thrombin-bound structures of native peptide fragments from the fifth EGF-like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF-like structure of an antiparallel beta-sheet, a novel structural motif observed for a bound peptide in protein-peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF-like domains, are unfavorably exposed in the complex of thrombin with the EGF-like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique beta-sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side-chain interactions within a specific environment may dictate the folding of bound peptides in protein-peptide complexes.     

Mapping the orientation of an antigenic peptide bound in the antigen binding groove of H-2Kb using a monoclonal antibody.

The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2Kb containing a defined peptide in the antigen cleft. We report that the binding surface area of a Kb specific monoclonal antibody (28-13-3s) includes residues in the alpha 1 (Gly56 and Glu58) and alpha 2 (Trp167) helices of Kb thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the alpha 1 helix.