Structural basis of constitutive c-Src kinase activity due to R175L and W118A mutations.

Cellular Src (c-Src) belongs to a non-receptor membrane-associated tyrosine kinase family that plays essential roles in cellular processes. Growing evidence suggests that R175L and W118A mutations in SH2/SH3 domains of c-Src functionally inactivate these domains leading to constitutive activation of kinase domain (KD). Here we modeled c-SrcR175L, c-SrcW118A and c-SrcW118A+R175L structures by inducing phosphorylation at Y416 or Y527, respectively to characterize the comparative dynamics in the active versus inactive states through molecular dynamics simulation assay. We observed more conformational readjustments in c-Srcopen than its close variants. In particular, C-terminal tail residues of c-SrcW118A-open and c-SrcW118A+R175L-open demonstrate significantly higher transitions. The cross-correlation analysis revealed an anticorrelation behavior in the motion of KD with respect to SH2, SH3 and the linker region of SrcW118A+R175L-open, while in c-SrcWT-open, SH2 and SH3 domains were anticorrelated, while KD and C-terminal tail motions were correlated. Due to these conformational differences, c-Src open forms exhibited lower interaction between pY527 and SH2 domain. Through detailed structural analysis, we observed a uniform myristate binding cavity in c-SrcWT-open, while the myristoyl pockets of mutant forms were deformed. We propose that constitutive activation of mutant Src forms may presumably be achieved by the prolonged membrane binding due to unusual conformations of C-terminal and myristoyl switch residues that may result in a higher dephosphorylation rate at pY527 in the myristoylated c-Src. Thus, our study establishes novel clues to decipher the constitutive activation status of c-Src in response to known mutations that may help in devising novel therapeutic strategies for cancer metastasis treatment.Communicated by Ramaswamy H. Sarma.

SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function.

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Autophosphorylation activates c-Src kinase through global structural rearrangements.

The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr416 using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations. We show that the initial steps of kinase activation involve large rearrangements in domain orientation. The kinase domain is highly dynamic and has strong cross-talk with the regulatory domains, which are displaced by autophosphorylation. Although the regulatory domains become more flexible and detach from the kinase domain because of autophosphorylation, the kinase domain gains rigidity, leading to stabilization of the ATP binding site and a 4-fold increase in enzymatic activity. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms.

High resolution crystal structure of the FAK FERM domain reveals new insights on the Druggability of tyrosine 397 and the Src SH3 binding site.

BACKGROUND: Focal Adhesion Kinase (FAK) is a major cancer drug target that is involved in numerous aspects of tumor progression and survival. While multiple research groups have developed ATP-competitive small molecule inhibitors that target the kinase enzyme, recent attention has been focused on the FAK FERM (Band 4.1, Ezrin, Radixin, Moesin) domain that contains key residue Y397 and contributes to many protein-protein interactions. Previous x-ray crystal structures of the FAK FERM domain gave conflicting results on the structure of the Y397 region and therefore the overall druggability. RESULTS: Here, we report the identification of a higher resolution crystal structure of the avian FAK FERM domain that shows conformational differences in Y397 and surrounding residues in the F1 lobe. In addition, we resolve the residues of the Src SH3 binding site, an area of the FERM domain that has previously shown limited electron density. CONCLUSIONS: These crystallographic data suggest that the Y397 region is highly dynamic and question the druggability of a putative pocket on the F1 lobe. In addition, new electron density data around the Src SH3 binding site provide structural insight on the FAK-Src activation cascade through a putative auto-inhibitory conformation.

The major ß-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo.

In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-ß-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the ß-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of ß-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the ß-catenin pathway process.

Benzo[a]pyrene activates an AhR/Src/ERK axis that contributes to CYP1A1 induction and stable DNA adducts formation in lung cells.

Benzo[a]pyrene (B[a]P), the most extensively studied carcinogen in cigarette smoke, has been regarded as a critical mediator of lung cancer. It is known that B[a]P-mediated Aryl hydrocarbon Receptor (AhR) activation stimulates the mitogen activated protein kinases (MAPK) signaling cascade in different cell models. MAPK pathway disturbances drive alterations in cellular processes, such as differentiation, proliferation, and apoptosis, and the disturbances may also modify the AhR pathway itself. However, MAPK involvement in B[a]P metabolic activation and toxicity in lung tissues is not well understood. Here, we used a non-transformed human bronchial epithelial lung cell line, BEAS-2B, to study the participation of ERK 1/2 kinases in the metabolic activation of B[a]P and in its related genotoxic effects. Our results indicate that B[a]P is not cytotoxic to BEAS-2B cells at relatively low concentrations, but it enhances CYP1A1 gene transcription and protein induction. Additionally, B[a]P promotes Src and ERK 1/2 phosphorylation. Accordingly, inhibition of both Src and ERK 1/2 phosphorylation decreases CYP1A1 protein induction, AhR nuclear translocation and production of B[a]P adducts. Together, these data suggest a crosstalk between AhR and the members of the MAPK pathway, ERK 1/2 mediated by Src kinase. This interaction is important for the adequate AhR pathway signaling that in turn induces transcription and protein induction of CYP1A1 and B[a]P-induced DNA damage in BEAS-2B cells.

Emergence of a Promising Lead Compound in the Treatment of Triple Negative Breast Cancer: An Insight into Conformational Features and Ligand Binding Landscape of c-Src Protein with UM-164.

UM-164, a potent Src/p38 inhibitor, is a promising lead compound for developing the first targeted therapeutic strategy against triple-negative breast cancer (TNBC). However, lack of understanding of conformational features of UM-164 in complex with Src serves a challenge in the rational design of novel Src dual inhibitors. Herein, we provide an in-depth insight into conformational features of Src-UM-164 using different computational approaches. This involved molecular dynamics (MD) simulation, principal component analysis (PCA), thermodynamics calculations, dynamic cross-correlation (DCCM) analysis, and hydrogen bond formation. Findings from this study revealed that (1) the binding of UM-164 to Src induces a more stable and compact conformation; (2) the binding of UM-164 results in increased correlation among the active site residue; (3) the presence of multiple phenyl rings and fluorinated phenyl group in UM-164 contributes to the steric effect; (4) a relatively high-binding free energy estimated for the Src-UM-164 system is affirmative of its experimental potency; (5) hydrophobic packing contributes significantly to the drug binding in Src-UM-164; and (6) observed increase in H-bond distance of interacting residue atoms and Dasatinib compared to UM-164. Findings from this study can serve as a baseline in the design of novel Src inhibitors with dual inhibitory properties.

A Ser75-to-Asp phospho-mimicking mutation in Src accelerates ageing-related loss of retinal ganglion cells in mice.

Src knockout mice show no detectable abnormalities in central nervous system (CNS) post-mitotic neurons, likely reflecting functional compensation by other Src family kinases. Cdk1- or Cdk5-dependent Ser75 phosphorylation in the amino-terminal Unique domain of Src, which shares no homology with other Src family kinases, regulates the stability of active Src. To clarify the roles of Src Ser75 phosphorylation in CNS neurons, we established two types of mutant mice with mutations in Src: phospho-mimicking Ser75Asp (SD) and non-phosphorylatable Ser75Ala (SA). In ageing SD/SD mice, retinal ganglion cell (RGC) number in whole retinas was significantly lower than that in young SD/SD mice in the absence of inflammation and elevated intraocular pressure, resembling the pathogenesis of progressive optic neuropathy. By contrast, SA/SA mice and wild-type (WT) mice exhibited no age-related RGC loss. The age-related retinal RGC number reduction was greater in the peripheral rather than the mid-peripheral region of the retina in SD/SD mice. Furthermore, Rho-associated kinase activity in whole retinas of ageing SD/SD mice was significantly higher than that in young SD/SD mice. These results suggest that Src regulates RGC survival during ageing in a manner that depends on Ser75 phosphorylation.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.

Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.

Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine.

BACKGROUND: C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. METHODS: We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. RESULTS: Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. CONCLUSIONS: SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.

Imatinib binding to human c-Src is coupled to inter-domain allostery and suggests a novel kinase inhibition strategy.

Imatinib (Gleevec), a non-receptor tyrosine kinase inhibitor (nRTKI), is one of the most successful anti-neoplastic drugs in clinical use. However, imatinib-resistant mutations are increasingly prevalent in patient tissues and driving development of novel imatinib analogs. We present a detailed study of the conformational dynamics, in the presence and absence of bound imatinib, for full-length human c-Src using hydrogen-deuterium exchange and mass spectrometry. Our results demonstrate that imatinib binding to the kinase domain effects dynamics of proline-rich or phosphorylated peptide ligand binding sites in distal c-Src SH3 and SH2 domains. These dynamic changes in functional regulatory sites, distal to the imatinib binding pocket, show similarities to structural transitions involved in kinase activation. These data also identify imatinib-sensitive, and imatinib-resistant, mutation sites. Thus, the current study identifies novel c-Src allosteric sites associated with imatinib binding and kinase activation and provide a framework for follow-on development of TKI binding modulators.

17ß-Estradiol induces odontoblastic differentiation via activation of the c-Src/MAPK pathway in human dental pulp cells.

The present study is aimed at investigating the effects of the exogenous estrogen 17ß-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.

Unraveling the molecular architecture of a G protein-coupled receptor/ß-arrestin/Erk module complex.

ß-arrestins serve as signaling scaffolds downstream of G protein-coupled receptors, and thus play a crucial role in a plethora of cellular processes. Although it is largely accepted that the ability of ß-arrestins to interact simultaneously with many protein partners is key in G protein-independent signaling of GPCRs, only the precise knowledge of these multimeric arrangements will allow a full understanding of the dynamics of these interactions and their functional consequences. However, current experimental procedures for the determination of the three-dimensional structures of protein-protein complexes are not well adapted to analyze these short-lived, multi-component assemblies. We propose a model of the receptor/ß-arrestin/Erk1 signaling module, which is consistent with most of the available experimental data. Moreover, for the ß-arrestin/Raf1 and the ß-arrestin/ERK interactions, we have used the model to design interfering peptides and shown that they compete with both partners, hereby demonstrating the validity of the predicted interaction regions.

The N2-Src neuronal splice variant of C-Src has altered SH3 domain ligand specificity and a higher constitutive activity than N1-Src.

N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also had a higher basal kinase activity than N1- and C-Src in vitro and in cells, which could be explained by weakened intramolecular interactions. Therefore, N2-Src is a highly active kinase that is likely to phosphorylate alternative substrates to C-Src in the brain.

The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src.

Regulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.

Intracellular cytoplasm-specific delivery of SH3 and SH2 domains of SLAP inhibits TcR-mediated signaling.

Signaling events triggered by T cell receptor (TcR) stimulation are important targets for the development of common therapeutics for various autoimmune diseases. SLAP is a negative regulator of TcR-mediated signaling cascade via targeting TcR zeta chain for degradation through recruiting the ubiquitin ligase c-Cbl. In this study, we generated a transducible form of SH3 and SH2 domains of SLAP (ctSLAPΔC) which can be specifically targeted to the cytoplasm of a cell. ctSLAPΔC inhibited tyrosine phosphorylation of signaling mediators such as ZAP-70 and LAT involved in T cell activation, and effectively suppressed transcriptional activity of NFAT and NFκB upon TcR stimulation. The transduced ctSLAPΔC in T cells blocked the secretion of T cell-specific cytokines such as IL-2, IFNγ, IL-17A, and IL-4 and induced the expression of CD69 and CD25 on effector T cells without influencing the cell viability. Inhibition of TcR-mediated signaling via SLAP blocked the differentiation of naïve T cells into Th1, Th2 or Treg cells with different sensitivity, suggesting that qualitative and quantitative intensity of TcR-mediated signaling in the context of polarizing cytokines environment may be a critical factor to determine the differentiation fate of naïve T cells. These results suggest that cytoplasm-specific transduction of the SH3 and SH2 domains of SLAP has a therapeutic potential of being an immunosuppressive reagent for the treatment of various autoimmune diseases.

Role of SRC-like adaptor protein (SLAP) in immune and malignant cell signaling.

SRC-like adaptor protein (SLAP) is an adaptor protein structurally similar to the SRC family protein kinases. Like SRC, SLAP contains an SH3 domain followed by an SH2 domain but the kinase domain has been replaced by a unique C-terminal region. SLAP is expressed in a variety of cell types. Current studies suggest that it regulates signaling of various cell surface receptors including the B cell receptor, the T cell receptor, cytokine receptors and receptor tyrosine kinases which are important regulator of immune and cancer cell signaling. SLAP targets receptors, or its associated components, by recruiting the ubiquitin machinery and thereby destabilizing signaling. SLAP directs receptors to ubiquitination-mediated degradation and controls receptors turnover as well as signaling. Thus, SLAP appears to be an important component in regulating signal transduction required for immune and malignant cells.

RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

Why ligand cross-reactivity is high within peptide recognition domain families? A case study on human c-Src SH3 domain.

Many important protein-protein interactions in eukaryotic signaling networks are mediated by peptide recognition domains (PRDs), which bind short linear sequence motifs in other proteins. However, high ligand cross-reactivity is observed within most PRD families, rendering a broad specificity for the family members. In the present study, we attempt to explore the molecular mechanism and physicochemical origin of PRD cross-reactivity. In the procedure, a structure-based method called atomic cross-nonbonded interaction analysis (ACNIA) is described to extract atomic-level nonbonded interaction information at domain-peptide interface and to correlate the information with peptide affinity based on a set of structure-solved, affinity-known protein-peptide complex samples compiled from numerous literatures and databases. The ACNIA-derived affinity predictor is tested rigorously with statistical validation approach, which is also demonstrated to be capable of perceiving slight structural change in the interface using three distinct panels of SH3-binding peptide data. Subsequently, with help of the affinity predictor we adopt the human c-Src SH3 domain, one of the most sophisticated PRDs, as a paradigm to investigate the ligand cross-reactivity within SH3 family. It is found that most of the family members have only few non-essential residue differences in their peptide-binding pockets, and thus exhibit a similar peptide recognition profile and high cross-reactivity. The cross-reactivity is even shared by different subclasses of SH3 domains. The findings suggest that inherent binding specificity is not the only factor to select appropriate binders for specific SH3 domains, and other aspects such as cellular context and the rest of the SH3-containing proteins may play important roles in reducing their ligand cross-reactivity.