Self-association and backbone dynamics of the hck SH2 domain in the free and phosphopeptide-complexed forms.
Decreased dynamic motion in the peptide backbone of proteins may accompany ligand binding and influence the thermodynamic and kinetic stability of the resulting complexes. We have investigated the diffusional behavior and backbone dynamics of the free and phosphopeptide (EPQpYEEIPIYL) complexed Hck SH2 domain using NMR spectroscopy. Both the free domain and its phosphopeptide complex self-associate at higher protein concentrations. Diffusional measurements and surface analysis indicate that charged side-chain groups are probably responsible for self-association. Higher order aggregation, such as trimer and tetramer, also occurs at elevated protein concentrations. Dynamic motion in the peptide backbone of Hck SH2 was determined from 15N relaxation data fit using extended model-free parameters. The rotational correlation time (taum) for uncomplexed Hck SH2 was 6.8 ns while taum for peptide-bound Hck SH2 was 7.6 ns. Generalized order parameters (S2) increased for most residues upon binding of the phosphopeptide, consistent with peptide binding restricting motion of the NH bond vectors on the picosecond time scale. These studies suggest that complexation increases internal order in Hck SH2 and that internal dynamic motions contribute to the activation of Src-family kinases in vivo.
Zhang
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W
The Eppley Institute for Research in Cancer and Allied Diseases, The University of Nebraska Medical Center, Omaha 68198-6805, USA.
Smithgall
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TE
Gmeiner
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WH
D006801:Humans; D006859:Hydrogen; D046911:Macromolecular Substances; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010748:Phosphopeptides / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D051572:Proto-Oncogene Proteins c-hck; D013816:Thermodynamics; D018909:src Homology Domains
D046911:Macromolecular Substances; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D006859:Hydrogen; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck