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Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, USA

Reprint requests to: Deborah S. Wuttke, 215 UCB, Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309-0215, USA; e-mail: Deborah.Wuttke{at}Colorado.Edu; fax: (303) 492-5894.

The N-terminal SH2 domain from the p85 subunit of phosphatidylinositol 3` kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like ¹H-¹⁵N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.

Keywords: Fragment complementation; fragment reconstitution; protein–protein interactions; protein folding; nuclear magnetic resonance spectroscopy; limited proteolysis

Abbreviations: SH2, Src homology 2 • NMR, nuclear magnetic resonance • HSQC, heteronuclear single quantum coherence • p85, N-terminal SH2 domain of the p85 • subunit of phosphatidylinositol 3` kinase • CD, circular dichroism • MALDI TOF, matrix assisted laser desorption ionization time of flight • DLS, dynamic light scattering • ACN, acetonitrile • GndHCl, guanidine hydrochloride • IPTG, isopropyl thiogalactoside • SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis • Fmoc, 9-fluorenylmethoxycarbonyl • HBTU, 2–(1-H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate • HOBt, N-hydroxybenzotriazole • DMF, N, N-dimethylformamide • DIEA, N, N-diisopropylethylamine

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