title: Structural comparison of the unstable drkN SH3 domain and a stable mutant.
abstract: The N-terminal SH3 domain of the Drosophila adapter protein Drk (drkN SH3 domain) is marginally stable (DeltaG(U) = 1 kcal/mol) and exists in equilibrium between folded and highly populated unfolded states. The single substitution T22G, however, completely stabilizes the protein (DeltaG(U) = 4.0 kcal/mol). To probe the causes of instability of the wild-type (WT) protein and the dramatic stabilization of the mutant, we determined and compared nuclear magnetic resonance structures of the folded WT and mutant drkN SH3 domains. Residual dipolar coupling (RDC) and carbonyl chemical-shift anisotropy (C'-CSA) restraints measured for the WT and T22G domains were used for calculating the structures. The structures for the WT and mutant are highly similar. Thr22 of the WT and Gly22 of the mutant are at the i + 2 position of the diverging, type-II beta-turn. Interestingly, not only Gly22 but also Thr22 successfully adopt an alpha(L) conformation, required at this position of the turn, despite the fact that positive phi values are energetically unfavorable and normally disallowed for threonine residues. Forcing the Thr22 residue into this unnatural conformation increases the free energy of the folded state of the WT domain relative to its T22G mutant. Evidence for residual helix formation in the diverging turn region has been previously reported for the unfolded state of the WT drkN SH3 domain, and this, in addition to other residual structure, has been proposed to play a role in decreasing the free energy of the unfolded state of the protein. Together these data provide evidence that both increasing the free energy of the folded state and decreasing the free energy of the unfolded state of the protein contribute to instability of the WT drkN SH3 domain.
authors.affiliation: The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada.
authors.forename: Irina
authors.initials: I
authors.lastname: Bezsonova
authors.forename: Alex
authors.initials: A
authors.lastname: Singer
authors.forename: Wing-Yiu
authors.initials: WY
authors.lastname: Choy
authors.forename: Martin
authors.initials: M
authors.lastname: Tollinger
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
mesh_terms: D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D018909:src Homology Domains / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D029721:Drosophila Proteins; C080530:drk protein, Drosophila