Ligand specificity modulated by prolyl imide bond Cis/Trans isomerization in the Itk SH2 domain: a quantitative NMR study

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) binds two separate ligands: a phosphotyrosine-containing peptide and the Itk Src homology 3 (SH3) domain. Binding specificity for these ligands is regulated via cis/trans isomerization of the Asn 286-Pro 287 imide bond in the Itk SH2 domain. In this study, we develop a novel method of analyzing chemical shift perturbation and cross-peak volumes to measure the affinities of both ligands for each SH2 conformer. We find that the cis imide bond containing SH2 conformer exhibits a 3.5-fold higher affinity for the Itk SH3 domain compared with binding of the trans conformer to the same ligand, while the trans conformer binds phosphopeptide with a 4-fold greater affinity than the cis-containing SH2 conformer. In addition to furthering the understanding of this system, the method presented here will be of general application in quantitatively determining the specificities of conformationally heterogeneous systems that use a molecular switch to regulate binding between multiple distinct ligands.     

Molecular recognition properties of FN3 monobodies that bind the Src SH3 domain

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.     

A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors

Background: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets.Results: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors.Conclusions: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.     

Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities

Introduction: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins. Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains. Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity.Results: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains. These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5. Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains. Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not. Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands.Conclusions: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Synthesis of 5-, 6- and 7-substituted-2-aminoquinolines as SH3 domain ligands

The Src homology 3 (SH3) domains are small protein-protein interaction domains that mediate a range of important biological processes and are considered valuable targets for the development of therapeutic agents. We have been developing 2-aminoquinolines as ligands for SH3 domains--so far the only reported examples of entirely small-molecule ligands for the SH3 domains. The highest affinity 2-aminoquinolines so far identified are 6-substituted compounds. In this article, the synthesis of several new 2-aminoquinolines, including 5-, 6- and 7-substituted compounds, for Tec SH3 domain ligand binding studies is presented. As a part of the synthetic investigation, the utility of different methods for the synthesis of 2-aminoquinolines was explored and potentially powerful methods were identified for the synthesis of 2-aminoquinolines with diverse functionality. Of the compounds prepared, the 5-substituted-2-aminoquinolines generally bound with similar affinities to unsubstituted 2-aminoquinoline, whilst the 7-substituted compounds generally bound with similar or lower affinity than unsubstituted 2-aminoquinoline. However, the 6-substituted-2-aminoquinolines generally bound with significantly higher affinity than unsubstituted 2-aminoquinoline. In addition, one 6-substituted-N-benzylated-2-aminoquinoline was also tested for SH3 binding and some evidence for the formation of additional contacts at other regions of the SH3 domain was found. These results provide new and useful SAR information that should greatly assist with the challenge of developing high affinity small-molecule ligands for the SH3 domains.     

Engineering temperature-sensitive SH3 domains.

BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks. SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences. RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive. Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature. Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins. Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells. CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo. Such mutants will be valuable in dissecting SH3-mediated signaling pathways. Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions.     

Protein microarray assay for the screening of SH3 domain interactions

Analysis of cellular signal transduction processes increasingly focuses on the systematic characterization of complete protein interaction networks. Understanding the interplay of signaling components enables insight into the molecular basis of diverse diseases such as cancer. This paves the way for the rational design of specific therapeutics. Protein interactions are often mediated by conserved modular domains, e.g., SH3-domains, which recognize proline-rich sequences in their cognate ligands. In the course of this study, different microarray formats (reactive silane monolayers and nitrocellulose on glass slides) and assay work flows were evaluated to develop a microarray based screening assay that permits the reliable identification of interactions between certain target proteins with a set of SH3 domains. Nine representative SH3 domains which were produced and purified as GST-fusion proteins were spotted on the microarray substrates and probed with two well-characterized ligands, the Nef protein from HIV-1 and the human protein Sam68. The best results from these low-density model arrays were obtained with nitrocellulose slides. We show that a straightforward and highly robust detection of ligand binding is achieved by staining with a fluorescently labeled antibody directed against the N-terminal His-tag attached to these proteins. The optimized assay protocol reported here allows for the identification of SH3-interactions with high reproducibility and adequate signal-to-background and signal-to-noise ratios, as well as the quantitative determination of relative binding affinities.     

A Src SH2 selective binding compound inhibits osteoclast-mediated resorption

BACKGROUND: The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS: AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS: These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.     

Co‐Evolutionary Fitness Landscapes for Sequence Design

Efficient and accurate models to predict the fitness of a sequence would be extremely valuable in protein design. We have explored the use of statistical potentials for the coevolutionary fitness landscape, extracted from known protein sequences, in conjunction with Monte Carlo simulations, as a tool for design. As proof of principle, we created a series of predicted high‐fitness sequences for three different protein folds, representative of different structural classes: the GA (all‐α) and GB (α/β) binding domains of streptococcal protein G, and an SH3 (all‐β) domain. We found that most of the designed proteins can fold stably to the target structure, and a structure for a representative of each for GA, GB and SH3 was determined. Several of our designed proteins were also able to bind to native ligands, in some cases with higher affinity than wild‐type. Thus, a search using a statistical fitness landscape is a remarkably effective tool for finding novel stable protein sequences.     

Combinatorial Synthesis and Multidimensional NMR Spectroscopy: An Approach to Understanding Protein–Ligand Interactions

Combinatorial chemistry is a laboratory emulation of natural recombination and selection processes. Strategies in this developing discipline involve the generation of diverse, molecular libraries through combinatorial synthesis and the selection of compounds that possess a desired property. Such approaches can facilitate the identification of ligands that bind to biological receptors, promoting our chemical understanding of cellular processes. This article illustrates that the coupling of combinatorial synthesis, multidimensional NMR spectroscopy, and biochemical methods has enhanced our understanding of a protein receptor used commonly in signal transduction, the Src Homology 3 (SH3) domain. This novel approach to studying molecular recognition has revealed a set of rules that govern SH3–ligand interactions, allowing models of receptor–ligand complexes to be constructed with only a knowledge of the polypeptide sequences. Combining combinatorial synthesis with structural methods provides a powerful new approach to understanding how proteins bind their ligands in general.     

Cyclic peptidyl inhibitors of Grb2 and tensin SH2 domains identified from combinatorial libraries

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. In this work, a novel method has been developed for the high-throughput synthesis, screening, and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic phosphotyrosyl peptide libraries containing randomized amino acid sequences and different ring sizes (theoretical diversity of 3.2 x 10(6)) were synthesized and screened against the SH2 domains of Grb2 and tensin. Potent, selective inhibitors were identified from the libraries and were generally more effective than the corresponding linear peptides. One of the inhibitors selected against the Grb2 SH2 domain inhibited human breast cancer cell growth and disrupted actin filaments. This method should be applicable to the development of cyclic peptidyl inhibitors against other protein domains, enzymes, and receptors.     

An analysis of the interactions between the Sem-5 SH3 domain and its ligands using molecular dynamics, free energy calculations, and sequence analysis.

The Src-homology-3 (SH3) domain of the Caenorhabditis elegans protein Sem-5 binds proline-rich sequences. It is reported that the SH3 domains broadly accept amide N-substituted residues instead of only recognizing prolines on the basis of side chain shape or rigidity. We have studied the interactions between Sem-5 and its ligands using molecular dynamics (MD), free energy calculations, and sequence analysis. Relative binding free energies, estimated by a method called MM/PBSA, between different substitutions at sites -1, 0, and +2 of the peptide are consistent with the experimental data. A new method to calculate atomic partial charges, AM1-BCC method, is also used in the binding free energy calculations for different N-substitutions at site -1. The results are very similar to those obtained from widely used RESP charges in the AMBER force field. AM1-BCC charges can be calculated more rapidly for any organic molecule than can the RESP charges. Therefore, their use can enable a broader and more efficient application of the MM/PBSA method in drug design. Examination of each component of the free energy leads to the construction of van der Waals interaction energy profiles for each ligand as well as for wild-type and mutant Sem-5 proteins. The profiles and free energy calculations indicate that the van der Waals interactions between the ligands and the receptor determine whether an N- or a Calpha-substituted residue is favored at each site. A VC value (defined as a product of the conservation percentage of each residue and its van der Waals interaction energy with the ligand) is used to identify several residues on the receptor that are critical for specificity and binding affinity. This VC value may have a potential use in identifying crucial residues for any ligand-protein or protein-protein system. Mutations at two of those crucial residues, N190 and N206, are examined. One mutation, N190I, is predicted to reduce the selectivity of the N-substituted residue at site -1 of the ligand and is shown to bind similarly with N- and Calpha-substituted residues at that site.     

Targeting metastable coiled-coil domains by computational design

Approximately 30% of eukaryotic genomes are predicted to encode partially unfolded proteins. Many of these unstructured domains contact multiple partners in short-lived interactions critical for cellular homeostasis. Understanding the functional implications of these transient binding events is a current challenge that could be addressed with designed peptide inhibitors. Most current protein design methodologies, however, target only structurally well-defined, stable structures. To address this limitation, we implemented a computational design strategy that alternates between a fixed backbone sequence search for binding specificity and structural optimization of the designed interfaces. We applied this method to create specific peptide inhibitors of the C-terminal metastable coiled-coil domain of the essential yeast septin Cdc12p. Specific binding of the designed sequences was demonstrated by circular dichroism and equilibrium ultracentrifugation. Our results validate computational methods to design specific peptide ligands to protein domains lacking intrinsic structural stability and set the stage for functional analysis of Cdc12p coiled coil function in vivo.     

Structural Investigations on the SH3b Domains of Clostridium perfringens Autolysin through NMR Spectroscopy and Structure Simulation Enlighten the Cell Wall Binding Function

Clostridium perfringens autolysin (CpAcp) is a peptidoglycan hydrolase associated with cell separation, division, and growth. It consists of a signal peptide, ten SH3b domains, and a catalytic domain. The structure and function mechanisms of the ten SH3bs related to cell wall peptidoglycan binding remain unclear. Here, the structures of CpAcp SH3bs were studied through NMR spectroscopy and structural simulation. The NMR structure of SH3b6 was determined at first, which adopts a typical β-barrel fold and has three potential ligand-binding pockets. The largest pocket containing eight conserved residues was suggested to bind with peptide ligand in a novel model. The structures of the other nine SH3bs were subsequently predicted to have a fold similar to SH3b6. Their ligand pockets are largely similar to those of SH3b6, although with varied size and morphology, except that SH3b1/2 display a third pocket markedly different from those in other SH3bs. Thus, it was supposed that SH3b3-10 possess similar ligand-binding ability, while SH3b1/2 have a different specificity and additional binding site for ligand. As an entirety, ten SH3bs confer a capacity for alternatively binding to various peptidoglycan sites in the cell wall. This study presents an initial insight into the structure and potential function of CpAcp SH3bs.     

Importance of CH/π hydrogen bonds in recognition of the core motif in proline-recognition domains: an ab initio fragment molecular orbital study

We examined CH/π hydrogen bonds in protein/ligand complexes involving at least one proline residue using the ab initio fragment molecular orbital (FMO) method and the program CHPI. FMO calculations were carried out at the Hartree–Fock (HF)/6‐31G*, HF/6‐31G**, second‐order Møller–Plesset perturbation (MP2)/6‐31G*, and MP2/6‐31G** levels for three Src homology 3 (SH3) domains and five proline‐recognition domains (PRDs) complexed with their corresponding ligand peptides. PRDs use a conserved set of aromatic residues to recognize proline‐rich sequences of specific ligands. Many CH/π hydrogen bonds were identified in these complexes. CH/π hydrogen bonds occurred, in particular, in the central part of the proline‐rich motifs. Our results suggest that CH/π hydrogen bonds are important in the recognition of SH3 and PRDs by their ligand peptides and play a vital role in the signal transduction system. Combined use of the FMO method and CHPI analysis is a valuable tool for the study of protein/protein and protein/ligand interactions and may be useful in rational drug design. © 2011 Wiley Periodicals, Inc. J Comput Chem 2011     

Probing the phosphopeptide specificities of protein tyrosine phosphatases,SH2 and PTB domains with combinatorial library methods

Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains. This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed.     

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site‐specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N‐terminally thiolated peptide hydrazides by hydrazone ligation, b) on‐surface native chemical ligation with self‐purified peptide thioesters, c) radical‐induced desulfurization, and d) a surface‐based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline‐rich ligands.     

Acquisition of Fyn-selective SH3 domain ligands via a combinatorial library strategy

A stepwise library-based strategy has been employed to acquire a potent ligand for the SH3 domain of Fyn, a Src kinase family member that plays a key role in T cell activation. The easily automated methodology is designed to identify potential interaction sites that circumscribe the protein/peptide binding region on the SH3 domain. The library protocol creates peptide/nonpeptide chimeras that are able to bind to these interaction sites that are otherwise inaccessible to natural amino acid residues. The peptide-derived lead and the Fyn-SH3 domain form a complex that exhibits a K(D) of 25 +/- 5 nM, approximately 1000-fold more potent than that displayed by the corresponding conventional peptide ligand. Furthermore, the lead ligand exhibits selectivity against SH3 domains derived from other Src kinases, in spite of a sequence identity of approximately 80%.