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R EVIEW ARTICLE

Implications of SH3 Domain Structure and Dynamics For Protein Regulation and Drug Design William H. Gmeiner* and David A. Horita Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157

Abstract SH3 Domains provide interesting targets for investigations of protein structure and dynamics because of their compact size and importance for signal transduction. The present review summarizes recent research investigating SH3 domain structure and dynamics, the discovery of novel SH3 domains, the role of SH3 domains in disease, and progress in targeting SH3 domains for the development of novel therapeutics. Particular emphasis is placed on the unfolding/refolding characteristics of SH3 domains and the potential importance of these processes for regulation of signal transduction. Index Entries: SH3 domain; NMR; Signal Transduction; Drug Design; Protein Dynamics.

SH3 domain structure apart from the study of other modules involved in signal transduction, e.g., SH2 domains, is the occurrence of unfolding/refolding processes that occur for SH3 domains on timescales that are reasonable for contributing to the regulation of biologically relevant processes (6,7). In this review, we will summarize the recent literature concerning SH3 domain participation in signal-transduction processes, and the structural and dynamical basis for SH3 domain-mediated regulation of signal transduction. Finally, we will summarize recent contributions to the literature that indicate that SH3 domains are important targets for drug design and discovery.

INTRODUCTION The previous 10 years have included major advances in understanding the nature of biological signal transduction (1,2) and how signaling processes are regulated by specific protein structural features (3). In particular, the structures of SH3 domains both as individual entities (4), and as portions of larger proteins involved in signal transduction (5), have been reported. Perhaps what has set research on * Author to whom all correspondence and reprint requests should be addressed. E-mail: bgmeiner@wfubmc. edu

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SH3 DOMAINS AS SIGNALING MODULES SH3 domains are examples of a modest number (~10) of well-characterized polypeptide domains that mediate intracellular signaling (8). Other domains that regulate intracellular signaling include SH2, PTB, PDZ, EVH1, WW, and PX domains (9). Each signaling domain is composed of 40–150 amino acids that is capable of folding into a 3D structure to create a noncatalytic binding pocket that binds short sequence motifs, typically consisting of 3–6 amino acids (3). The relatively small number of signaling domains with each having defined rules for ligand interaction is consistent with the existence of a “protein recognition code” analogous to the genetic code that governs protein–protein interactions mediated by signaling modules (9).

MECHANISMS OF SIGNALING The modular nature of SH domains, and other signaling domains, has contributed to a simple depiction of essentially linear signaltransduction cascades (e.g., Ras-Raf-MAPK) mediated by signaling proteins with component signaling modules (10). Under this scenario, signals are transduced in a linear cascade by a series of individual protein-protein interactions each involving one type of signaling domain (e.g., SH3), and one type of recognition motif (e.g., PxxP). These protein– protein interactions stimulate conformational changes, phosphorylation, or other post-translational modifications, which, in turn, allows the signaling cascade to proceed to the next step, ultimately resulting in a nuclear event (e.g., transcriptional activation). This simplistic model is deficient in at least three aspects. First, signaling domains may be involved in any one of a number of possible interactions at any given instant, thus cascades are rarely unique, linear pathways (1). In fact, mechanisms of signaling must be combinatorial in nature because of a limited number of gene products that

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Gmeiner and Horita transduce specific signals in different cell types (11). Second, signaling domains are frequently found in tandem or higher multiples (12). Multiple signaling domains in a protein may result in either positive or negative cooperativity in signaling. Further, adjacent signaling domains may physically interact and modulate signal-transducing capability (i.e., the bead on a string model may be inadequate in some instances; 13). Third, signaling modules are dynamic entities, and signal transduction depends not only on structural interactions, but also on the duration of the binding interaction. The dynamics of SH3 domains, and the implications of domain metastability for signaling, will be discussed in greater detail below. Thus, a signaling domain component of a signaling protein can potentially participate in any one of several possible interactions each of which contributes to different, perhaps even conflicting, signaling pathways (1,2). The actual protein–protein interactions that occur depends on the concentrations of potential binding partners and the relative free energies associated with these diverse interactions.

SH3 DOMAIN STRUCTURE SH3-mediated signaling processes are essentially driven by the recognition of polyprolineII helices by SH3 domain structures. The structures of SH3 domains were first reported in 1992 five β-strands termed βA–βE and a single turn of 310 helix (Fig. 1A; 16). The first two β-strands (βA–βB) are separated by a loop region (the RT-loop: named for conserved Arg and Thr residues), which itself has been observed to have substantial β-sheet character (17). The second and third β-strands (βB – βC) are separated by the n-Src loop, named for its substantially greater length in Src from neuronal tissue. The third and fourth β-strands are separated by the distal loop, while the fourth and fifth β-strands are separated by a stretch of 310 helix (Fig. 1B). The basic topology of the SH3 domain fold involves formation of two, triple-stranded, antiparallel β-sheets (βE, βA, βB

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Fig. 1. (A) Depiction of the typical secondary structure for SH3 domains, and three-dimensional structure of SH3 domains. The tertiary fold involves formation of two β-sheets (βE, βA, βB and βB, βC, βD) that form at right angles to one another. (B) The structure shown is of Hck SH3 (4,61).

and βB, βC, βD) that form at right angles relative to one another creating a hydrophobic pocket that binds polyproline helical peptides with moderate to high affinity (see following section). The compact size, relatively high solubility, and biological relevance of SH3 domains have made these signaling modules popular targets for nuclear magnetic resonance (NMR) and X-ray structural studies over the past decade. SH3 domains continue to serve as useful model systems for developing novel struc-

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tural tools, such as the application of solidstate Cross-Polarization Magic Angle Spinning (CP-MAS) NMR spectroscopy (18).

LIGAND RECOGNITION The structural basis for the binding of SH3 domains with proteins having an exposed PxxP motif has been elucidated (19–21). The PxxP motif adopts a polyproline type II (PPII) sec-

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Fig. 2. Depiction of the ligand-binding surface of PPII-helical peptides, or regions of proteins, that are recognized by SH3 domains. PPII-helices may bind SH3 domain in either the (+) or (–) orientation, depending on the relative position of the positively charged amino acid (R), which interacts with an acidic residue present in the RT-loop of nearly all SH3 domains. The (+) orientation is shown in (A) and the (–) orientation in (B).

ondary structure. In the PPII helix, exactly three residues constitute one left-handed helical turn (Fig. 2). Thus, the two prolines are adjacent to one another along one side of the helix (22). The intervening residues (xx in PxxP) often are also prolines, and the inclusion of additional prolines may stabilize the PPII helical conformation. Each of the prolines provides important hydrophobic contacts with the binding surface of the SH3 domain. The SH3 domain binding interface is composed of three pockets, two of which are formed by a series of mainly aromatic residues that are highly conserved among

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Gmeiner and Horita diverse SH3 domains. These two pockets bind xP elements in the PxxP recognition motif (23). The PxxP motif has pseudo-symmetry, and could potentially bind to the two binding pockets of a cognate SH3 domain in two orientations (Fig. 2). The placement of an aliphatic amino acid (θ) either adjacent to the PxxPmotif (“plus”; NH2-θPxxP-COOH), or within it (“minus”; NH2-PxθP-COOH), confers some degree of polarity to the PPII-helical ligand (Fig. 2). In most SH3 ligands, the major determinant of polarity is a basic amino acid (generally Arg) that occurs either three residues before (NH2RxθPxxP-COOH) or two residues after (NH2PxθPxR-COOH) the conserved PxxP motif. Ligands with the basic amino acid N-terminal to the PxxP motif are designated “plus” while those with the basic amino acid C-terminal are designated “minus” ligands. The specificity of the ligand orientation (plus or minus) with respect to the SH3 domain derives primarily from the presence of an acidic residue in the RTloop of most SH3 domains, which forms a saltbridge with Arg. The formation of this salt bridge enforces opposite polarity for the two classes of ligands, relative to the SH3 domain. The majority of SH3-binding ligands studied to date are readily classified as either Type I or Type II ligands. For example, the amino acid sequences containing the PPII-helical region of Nef proteins from different viral strains all conform to the “minus” orientation for the SH3 ligand. A number of ligand-SH3 interactions, however, cannot be classified according to canonical binding interactions. For example, Horita and coworkers recently demonstrated that the PPII region of bGAP, which lacks a basic residue, interacted with the SH3 domain of human Hck with the ligand in a “minus” orientation (4). Distinct binding motifs may occur within phylogenetically distinct subfamilies within the SH3 family. For example, a PxxDY consensus sequence has been shown to be essential for binding to the SH3 domain of Eps8 (24). The X-ray structure of the SH3 domain from Eps8 unexpectedly revealed an intertwined dimer. Surprisingly this grossly rearranged dimeric structure resulted in half-

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SH3 Domain Structure dimers that retained structurally conserved SH3 folds whose central regions superimposed on the structures of more typical monomeric SH3 domains (25).

SH3 DOMAINS AND DISEASE SH3 domains are found in all eukaryotes, including yeasts, suggesting a longer evolutionary history and a more general function than SH2 domains (11). Domains with structures similar to SH3 domains have been identified in bacteria (26). A recent PSI-BLAST search (27) revealed the N-terminus of the P60 invasion protein from Listeria grayi had high homology to the eukaryotic SH3 domain from the tyrosine kinase from the Pacific eel ray (28). Threading calculations and model building provided convincing evidence that the Nterminus of the P60 invasion protein had an SH3 fold. P60 is important for bacterial invasion of epithelial cells (29), and survival within the host cell (30). Infection of host eukaryotic cells by most pathogenic bacteria is accompanied by tyrosine phosphorylation. Inhibition of tyrosine phosphorylation impairs infection (31). SH3 domains have been proposed to function in promoting survival of a bacterial pathogen within the invaded cell by modulating pathways controlled by SH3 domains, or by promoting invasion by binding to receptors in eukaryotic cells (28). SH3-like domains have also recently been characterized in the bacteria Thermotoga maritima (32) and Corynebacterium diphtheriae (33). In the latter instance, the SH3like module binds iron, suggesting that SH3like modules in prokaryotes may have diverse functions from those so far identified for these domains in eukaryotes. It is likely that genomic analyses will reveal SH3-like domains are widespread in prokaryotes. The putative role of bacterial SH3 domains in bacterial pathogenesis described by Whisstock and Lesk for Listeria grayi is, to some degree, the converse of the established role of the HIV viral protein Nef interacting with host cell SH3 domains to mediate viral pathogene-

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131 sis. Nef is a 27–34 kD myristoylated protein unique to primate lentiviruses such as HIV and SIV (22). Nef is required for high-titer replication of SIV and for induction of AIDS in rhesus monkeys (34). Viruses defective in the nef gene have been isolated from some humans who are experiencing long-term nonprogressive HIV-1 infection (35), including a HIV-1-infected longterm nonprogressing mother-child pair (36). Nef has no known catalytic activity and likely functions through interactions with cellular proteins involved in cellular activation and signaling, in part through SH3-mediated interactions involving a PxxP motif on Nef. Mutational studies have demonstrated the functional importance of the PxxP SH3-binding motif of Nef to viral infectivity and replication, and mutations to the PxxP motif of Nef also have a modest effect on CD4 downregulation (37). Nef has been shown to associate with Mitogen-activated protein kinase (MAPK) and a Nef-associated Ser/Thr kinase (NAK) (38,39). While NAK and related kinases lack SH3 domains, the interaction with Nef requires SH3 domain binding capability, and thus is probably mediated by an as yet unidentified protein with an SH3 domain. Nef also downregulates class I major histocompatibility complexes (MHC) and this activity of Nef also requires the SH3 domain-binding surface (40). Although the pathobiology of HIV infection and AIDS progression mediated by Nef is complex, an important component of Nefmediated activity apparently results from interactions between Nef and the SH3 domains of Src-family protein tyrosine kinases (PTKs). The SH3 domains of Lyn and Hck bind tightly to the PxxP-motif in Nef. Hck is mainly expressed in hematopoietic cells, especially macrophages, and macrophages may play an important role in HIV infection and in the development of AIDS (41). The affinity of the Nef/Hck-SH3 interaction has a KD of 0.2 µM, representing the tightest SH3/ligand interaction yet reported (42,43). Mutagenesis studies have revealed the structural basis for the very high affinity of Nef for the SH3 domain of Hck is a result of

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132 a hydrophobic binding pocket in the RT-loop of Hck SH3 (42). This hydrophobic pocket does not occur in the RT-loops of SH3 domains from Src-family PTKs other than Hck and Lyn (44). The diverse roles of SH3 domains in signaling processes is exemplified by the interactions of Src-family PTKs with Nef. SH3-mediated binding of Nef activates Hck, and in an appropriate cell system can result in cellular transformation (45). In contrast, Nef interferes with the catalytic activities of Lck, which has identical domain architecture to Hck (46). Thus, interactions between Nef and different Src-family PTKs result in divergent biological effects despite their sequence and structural similarities. These results exemplify the importance of structural and dynamical details of protein signaling modules in determining the mediation of signaling pathways. At present, the structural (and dynamical) details responsible for the biological activities of Src-family PTKs in response to Nef binding remain incompletely characterized. Additional studies may be insightful in elucidating fundamental rules of signal transduction involving SH3 domains, and in understanding the pathobiology of HIV.

SH3 REGULATION OF SRC-FAMILY PTKS Activation of Src-family PTKs can result in cellular transformation, and thus these proteins, together with the homologous protein Abl, represent a class of proteins that link SH3mediated signal transduction to the development of malignancy (47). Src-family PTKs consist of six domains (Fig. 3; 8): 1. An N-terminal region containing sites of myristylation, and in some cases, palmitylation. These lipid modifications are responsible for targeting Src-family PTKs to the plasma or intracellular membrane. 2. A unique domain consisting of 30–70 amino acids. Little sequence homology exists among Src-family PTKs in this region, and its biological significance is unclear.

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Gmeiner and Horita

Fig. 3. (A) Domain architecture of Src-family PTKs. The N-terminal domain (N) contains sites for post-translational modifications that anchor these proteins to the plasma membrane, while the function of the unique domain (U) is not clear. The SH3 and SH2 domains are involved in negative regulation of the kinase domain (SH1) and substrate recruitment. The C-terminal domain contains a phosphotyrosine (pY) that bind intramolecularly to SH2 domains in the negatively-regulated state. (B) Depiction of the domain architecture in the negatively-regulated state. (C) Depiction of the domain architecture when bound to a substrate. Substrates have either PPII-helical regions that interact with SH3 domains and/or phosphotyrosine that interacts with the SH2 domain.

3. An SH3 domain consisting of approx 60 amino acids. SH3 domains bind to prolinerich ligands that can adopt a left-handed helical conformation. SH3 domains contribute to substrate recognition, membrane localization, and negative regulation of kinase activity. 4. An SH2 domain consisting of approx 100 amino acids. SH2 domains bind tightly to

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Fig. 4. Structure of the regulatory domains (SH3 and SH2) and kinase domain of Hck (48) in the negatively regulated state in which the SH3 domain binds to the PPII-helical region in the linker region (L2K) that connects SH2 and the kinase domain.

specific sequences in proteins that contain a phosphotyrosine (pY) residue. 5. A kinase (SH1) domain that is responsible for the phosphorylation of tyrosine residues of substrate proteins. The general features of kinase domains are conserved among a large number of proteins that have kinase activity including all members of the Srcfamily PTKs. 6. A C-terminal tail with a negative-regulatory tyrosine residue. Interaction between pY in the C-terminal tail and the SH2 domain play a central role in the negative regulation of Src family kinase activity. The X-ray structures of the regulatory and kinase domains of Hck and c-Src clarified the general features of negative regulation for Srcfamily PTKs (Fig. 4; 48,49). These structures

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confirmed that intramolecular association between the SH2 domain and the tail region that is C-terminal to the kinase occurs, and plays an important role in deactivation of the kinase. Phosphorylation of the conserved tyrosine residue in the C-terminal tail region (Tyr 527 in Src; Tyr 501 in Hck) in the proteins studied by X-ray crystallography was accomplished using Csk (for C-terminal Src kinase), a distinct regulatory kinase that phosphorylates these residues in vivo. The importance of the SH2-pY527 interaction for kinase deactivation was previously demonstrated in studies showing the kinase activity of c-Src was increased more than 10-fold when its tail region Tyr residue (Tyr 527) was mutated to Phe (Y527F mutant). This Src mutant produced a transformed phenotype when introduced into rodent fibroblasts, indicating the important

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134 role of the Csk site in the negative regulation of Src kinase activity in living cells (50). The analogous tail mutant of Hck (Y501F) exhibited potent transforming activity in fibroblasts, while the wild-type protein was nontransforming (45). Conversely, upon mutating the residues in the vicinity of Tyr 501 in Hck to pYEEI, the sequence with the highest affinity for Hck SH2 (51), the kinase cannot be activated by exogenous SH2-binding ligands (52). These results demonstrate the subtle interactions that are responsible for kinase activation and signal transduction in Src-family PTKs, and other signaling molecules. SH3 domains had been previously observed to contribute to negative regulation of Src-family PTKs, however the structural basis for this contribution had not been discerned prior to publication of the X-ray structures of c-Src and Hck. In the X-ray structures for both Hck and cSrc, the SH3 domain was intramolecularly associated with the linker region between the SH2 domain and the kinase domain, L2K (Fig. 4). The L2K region of each protein adopted a lefthanded polyproline II helix structure observed previously to bind to SH3 domains, a surprising observation since the L2K region of neither protein is proline-rich (two prolines occur in L2K of Hck, one in c-Src). Mutations in the c-Src SH3 domain that activate the kinase are located at the SH3-L2k interface. Accumulation of point mutations in Src-family PTK SH3 domains activates the kinase, and leads to cellular transformation, by relaxing the interaction between the SH3 domain and the linker region (53). Interestingly, binding of Nef to Hck SH3 activated the kinase even when the tail region of Hck was mutated to pYEEI, suggesting a hierarchy of interactions in which SH3 binding events activate the kinase even when exogenous SH2 binding ligands are incapable of doing so (52). The mechanism by which the kinase domain of Src-family PTKs is regulated by SH2 and SH3 ligand binding remains an active area of research (52). Examination of the X-ray structures of Hck and c-Src revealed neither the SH2 nor the SH3 domain sterically occluded the

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Gmeiner and Horita active site of the kinase domain when engaged in intramolecular binding. Rather, the inhibition of kinase activity was indirect. The N- and C-lobes of the kinase domain require a conformation for activity in which the γ-phosphate of ATP is aligned with the substrate acceptor group. In this ‘active’ conformation, Glu 310 in helix αC interacts with the divalent metal ions associated with ATP. In the ‘inactive’ conformation, intramolecular binding of SH3 stabilized L2K in the PPII conformation, resulting in interactions between L2K and the C-terminal end of helix αC in the N-lobe of the kinase domain. Helix αC also interacted with the activation segment in the C-lobe of the kinase domain, and both interactions served to move Glu 310 in helix αC away from the ATP binding site. The ‘active’ conformation of the kinase was adopted upon autophosphorylation at Tyr 416 of helix αC and/or upon disengagement of L2k from SH3. Autophosphorylation at Tyr 416 resulted in hydrogen-bond formation between pTyr 416 and Arg 385 in the activation segment, and this, in turn, reduced interaction between the activation segment and helix αC. Mutation of Tyr 416 to Ala substantially increased the Km and decreased the Vmax for Hck (52). Disengagement of L2k from SH3 may increase the flexibility of L2k, thus reducing its interaction with helix αC. The diverse and subtle binding interactions involving SH3 domains in Src-family PTKs demonstrate the complexity of intervening in SH3-mediated processes for the treatment of disease.

DYNAMICS OF REGULATORY DOMAINS Recent studies using NMR spectroscopy, mass spectrometry, molecular dynamics simulations, and other biophysical methods have revealed that isolated SH2, and especially SH3 domains, undergo significant dynamic motion. The N-terminal SH3 domain of the Drosophila protein Drk existed as an equilibrium mixture of folded and unfolded forms in solution under conditions that were nearly physiological

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SH3 Domain Structure (54,55). The rate of interconversion between the folded and unfolded states for Drk SH3 was slow on the timescale for observation using NMR spectroscopy, and NMR spectroscopy was used by Forman-Kay and co-workers to study protein folding in SH3 domains. An equilibrium between folded and unfolded states was also observed for the N-terminal SH3 domain of Grb2, the mammalian homolog of Drk (56). The unfolded state of Drk SH3 is structurally distinct from the guanidinium denatured state of the domain (57). Deuterium labeling in this system permitted detection of long-range nuclear Ovehauser effect interactions (NOEs) between amide protons that indicated that the unfolded state consisted, in part, of compact conformations that resembled the folded state. NMR studies on the α-spectrin SH3 domain revealed the presence of unfolded conformers whose residual structure bears some resemblance to the structure of the folding transition state for this domain (58). Likewise, thermodynamic studies of the α-spectrin SH3 domain revealed the transition state was similar to the folded state with regards to changes in heat capacity and entropy, indicating a high degree of compactness and order in the transition state (59). Molecular dynamics simulations of Src SH3 unfolding revealed a hierarchy of protein unfolding that was analogous to experimental studies of the folding of Src SH3 (60), while molecular dynamics simulations together with NMR relaxation experiments were used to characterize rapid molecular motions in Hck SH3 (61). These studies, collectively, indicate that SH3 domains are inherently unstable, however the unfolded states resemble intact SH3 domains in many of their biophysical, and even structural, properties (62). This inherent instability, together with retention of many structural characteristics of the intact domain in the unfolded state, is consistent with SH3 domain unfolding/refolding having a significant role in the regulation of protein activity in vivo. Engineered SH3 domains with decreased structural stability have been proposed as a means to study protein-protein interactions in vivo (63), while mutational studies have iden-

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135 tified interactions that are important for domain stability (64). Our laboratories, in collaboration with Professor David L. Smith, have engaged in characterizing the unfolding dynamics of the Hck SH3 domain using mass spectrometry and NMR spectroscopy (6,7,65). Analysis of proteolytic fragments of Hck SH3 by mass spectroscopy following deuterium exchange into the intact Hck SH3 domain indicated a cooperative unfolding event involving 24–61% of the domain (6). This unfolding process occurred with a half-life of approx 20 min. Correlation of 2HEx-MS rates and isotope patterns revealed cooperative unfolding occurred in several regions of the domain, including the C-terminal half of the RT-loop and a β-sheet flanking the binding site. Binding of a prolyl-rich segment from the HIV protein Nef slowed unfolding by a factor of three. Further analysis yielded a KD of 25 µM for the Nef peptide, in good agreement with published values for this interaction measured using surface plasmon resonance (SPR) (42). These results are consistent with an inherent flexibility in the SH3 domain assisting interconversion of the closed, intramolecularly ligated state and the open, active state of Src family kinases. This type of previously undetectable slow unfolding process may provide the basis for previously undescribed mechanisms in which kinetics of local unfolding combines with thermodynamics to regulate enzymatic activity. For example, dynamic motion of Hck SH3 to release the domain from its interaction with [L2k] may be the first step in the activation of Hck that occurs prior to binding the HIV protein, Nef. NMR studies from our laboratory (66–71), and others (72,73) have indicated that the SH2 domain from Hck undergoes more limited dynamic motion than the unfolding that is observed for Hck SH3. Knowledge of the changes in dynamic motion of the peptide backbone that accompany ligand binding are especially important for understanding the molecular interactions that are responsible for activation of Src-family kinases in vivo (74–78). Our laboratory solved the structure of the SH2

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136 domain for Hck using NMR spectroscopy (70). We also described the diffusional behavior and the backbone dynamics of the isolated Hck SH2 domain (71). In both free and phosphopeptide complexed forms, not only rapid (ps) site-specific internal motions in the SH2 domain were observed, but also site-specific intermediate time-scale motions (2–5 ns) were observed across almost the entire backbone. Complexation of Hck SH2 with a high-affinity phosphopeptide (pYEEI) reduced the dynamic motion of the peptide backbone. These studies are consistent with complexation of Hck SH2 resulting in a more ordered structure implying that ligand release is entropically favored by increased internal motion in the SH2 domain. These studies demonstrated that complexation increased internal order in Hck SH2, and suggested that internal dynamic motions contribute to the activation of Src-family kinases in vivo by providing a thermodynamic driving force for release of the phosphotyrosine-containing tail region.

IMPLICATIONS FOR DRUG DESIGN Targeting signal-transduction cascades is a recognized approach for drug-discovery research (79). The importance of SH3 domains in mediating critical signaling processes that result in malignancies and other disorders has resulted in attempts to engineer drugs that would interfere in SH3-mediated processes (80). Although only a modest percentage of the research on the design and synthesis of ligands that interfere in SH3-mediated signaling processes has been published, the research reported has yielded some interesting new concepts in drug design, as well as application of drug-discovery technologies widely used in pursuit of diverse molecular targets. For example, the Schreiber laboratory has used combinatorial approaches to discover ligands for SH3 domains (81). A twist in the application of combinatorial methodologies to drug discovery was applied by Schumacher et al., who used an SH3 domain composed of L-amino acids to

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Gmeiner and Horita search for peptidic ligands composed of Damino acids (82). This approach allowed these researchers to define peptide substrates composed of L-amino acids to target the native SH3 domain (82). Other approaches have utilized organic synthesis to produce SH3 binding ligands that structurally resemble the PPII-helical peptide that is exposed preformed on the surface of proteins recognized by SH3 domains (83). Mutagenesis of known sequences was used to create peptides with high affinity and selectivity for the Crk family of adaptor and related proteins (84,85). These studies have also focused on identifying SH3 binding motifs that are membrane-permeable. Recognition that SH3 domains often appear in tandem with SH2 domains and that simultaneous targeting of SH3 and SH2 domains could result in ligands with very high affinity and specificity for specific signaling proteins led Cowburn and coworkers to develop consolidated ligands (86). These ligands have also proven useful in establishing the flexibility inherent in many contiguous SH3-SH2 constructs. Although SH3 domains are less well-developed than SH2 domains as targets for therapeutic development (87), their widespread occurrence in diverse biological processes will make SH3 domains the focus of drug-discovery research efforts for many years to come.

SUMMARY SH3 domains continue to provide interesting targets to explore concepts in protein folding, structure determination, interdomain regulation of signal-transduction cascades, and drug design. SH3 domains have now been found in prokaryotes as well as eukaryotes, and to regulate more diverse biological processes than previously realized. Continued research will provide additional insight into how SH3 domain structure and dynamics regulate intracellular signal-transduction cascades, and provide new challenges for the design of molecules to intervene in SH3-mediated processes.

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ACKNOWLEDGMENTS This work was supported by NIH-NCI CA60612, and Comprehensive Cancer Center of Wake Forest University, NIH P30 CA12197CCCWFU. Thanks to Christina Peklak for assembling references.

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