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Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

Department of Human Biological Chemistry and Genetics, and Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA

Reprint requests to: Vincent J. Hilser, University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, TX 77555, USA; e-mail: vince{at}hbcg.utmb.edu; fax: (409) 747-6816.

We report the effects of peptide binding on the ¹⁵N relaxation rates and chemical shifts of the C-SH3 of Sem-5. ¹⁵N spin-lattice relaxation time (T₁), spin-spin relaxation time (T₂), and {¹H}-¹⁵N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S² > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the ¹⁵N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.

Keywords: Sem-5; SH3; ¹⁵N relaxation; NMR; backbone dynamics; order parameter; polyproline peptide; ligand binding

Abbreviations: SH3, src-homology domain 3 • C-SH3, C-terminal SH3 domain • Sos, Son of Sevenless • NMR, nuclear magnetic resonance • 2D, two-dimensional • NOE, nuclear Overhauser effect • ITC, isothermal titration calorimetry • HSQC, heteronuclear single quantum coherence • ppm, parts per million • PDB, Protein Data Bank

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