Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase 1 1Edited by P. E. Wright

Bradshaw, J. Michael; Mitaxov, V; Waksman, Gabriel

doi:10.1006/jmbi.1999.3190

Cited by 140 publications

(150 citation statements)

References 46 publications

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“…Mutation of Lys 200 to Ala in the Src SH2 domain resulted in a 7-fold reduction in binding affinity for GpYEEI (32). In a recent computational analysis, the ␤D3 position is identified to be an energetically important residue in the function of a large num- Tyr 169 Thr 179 Ile 180 Ile 203 ber of SH2 domains, such as Hck, Lck, Src, Fyn, SHPTP2 N, Grb2, SAP, p85a C, and so forth (45).…”

Section: Discussionmentioning

confidence: 99%

Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

Ayrapetov¹,

Nam

et al. 2005

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

Ayrapetov¹,

Nam

et al. 2005

Journal of Biological Chemistry

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“…SH2 domains are highly conserved noncatalytic proteins of Ϸ100-aa residues, which can bind phosphotyrosine-containing polypeptide sequences with high affinity and specificity. They are found in a wide variety of intracellular signal transduction pathways involving tyrosine kinases (18)(19)(20)(21)(22), and inappropriate cellular signaling caused by malfunctions of SH2-mediated process has been linked to many pathologic conditions (e.g., cancer, autoimmune diseases, asthma, allergies, etc.). SH2 domains are highly selective toward the sequence phosphotyrosine-Glu-Glu-Ile (pYEEI) (23), with dissociation constants ranging from micromolar to nanomolar ranges (19,24; for a review, see ref.…”

mentioning

confidence: 99%

Calculation of absolute protein–ligand binding free energy from computer simulations

Woo

Roux²

2005

Proc. Natl. Acad. Sci. U.S.A.

627

896

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A general methodology for calculating the equilibrium binding constant of a flexible ligand to a protein receptor is formulated on the basis of potentials of mean force. The overall process is decomposed into several stages that can be computed separately: the free ligand in the bulk is first restrained into the conformation it adopts in the bound state, position, and orientation by applying biasing potentials, then it is translated into the binding site, where it is released completely. The conformational restraining potential is based on the root-mean-square deviation of the peptide coordinates relative to its average conformation in the bound complex. Free energy contributions from each stage are calculated by means of free energy perturbation potential of mean force techniques by using appropriate order parameters. The present approach avoids the need to decouple the ligand from its surrounding (bulk solvent and receptor protein) as is traditionally performed in the doubledecoupling scheme. It is believed that the present formulation will be particularly useful when the solvation free energy of the ligand is very large. As an application, the equilibrium binding constant of the phosphotyrosine peptide pYEEI to the Src homology 2 domain of human Lck has been calculated. The results are in good agreement with experimental values. Approaches at different levels of complexity and sophistication have been used to calculate binding free energies in complex biomolecular systems. Screening of large molecular databases of compounds to identify potential lead drug molecules typically relies on very simplified scoring schemes to achieve the needed efficiency (6). The binding free energy may be estimated on the basis of a continuum solvent approximation assuming quadratic fluctuations around a unique configuration (7,8). The Molecular Mechanics͞ Poisson-Boltzmann (PB) and Surface Area (MM͞PB-SA) method is a popular approach that relies on a mixed scheme combining configurations sampled from molecular dynamics (MD) simulations with explicit solvent, together with free energy estimators based on an implicit continuum solvent model (9). MM͞PB-SA shares some similarities with the linear interaction energy method, which also uses averages calculated from explicit solvent simulations within a linear response framework (10). Despite their usefulness, such approximate schemes can be limited, and how to improve the results is unclear because they do not offer a rigorous route to compute the equilibrium binding constant.In principle, treatments based on MD free energy perturbation (FEP) simulations with explicit solvent molecules offer the most powerful and promising approach to estimate the binding free energies of ligands to macromolecules (11). Nonetheless, although previous studies have provided many of the fundamental elements necessary for the calculation of binding free energy by means of MD (12-17), the computations so far have been limited mostly to fairly small and rigid ligands [e.g., rare gas atom (12, 15), water (14)...

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“…When bound to tyrosine phosphorylated ligands, C185 lies in close proximity to the phosphate group within the binding pocket, where the intrinsic repulsive nature of the deprotonated C185 facilitates release of pY527 from the SH2 domain, assisting in relieving the kinase from the autoinhibited state (Bradshaw et al, 1999). Our data provides an additional and alternative function for Src C185 in docking to cortactin, where cystine bonding to C112 or C246 mediates association with activated Src.…”

Section: Cystine Bonding Mediates Src Sh2 Binding To Cortactinmentioning

confidence: 82%

Src binds cortactin through an SH2 domain cystine-mediated linkage

Evans

Ammer

Jett

et al. 2012

Journal of Cell Science

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SummaryTyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actinbased motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Srccortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

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Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase 1 1Edited by P. E. Wright

Cited by 140 publications

References 46 publications

Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

Calculation of absolute protein–ligand binding free energy from computer simulations

Src binds cortactin through an SH2 domain cystine-mediated linkage

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