Role of the conserved acidic residue Asp21 in the structure of phosphatidylinositol 3-kinase Src homology 3 domain: Circular dichroism and nuclear magnetic resonance studies

To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue As p21 of the phosphatidylinositol 3-kinase (PI3K) SH3 domain, structural chan ges induced by the D21N mutation (Asp21 --> Asn) were examined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies, En the previous study, we demonstrated that environmental alterations occurred at the side chains of Trp55 and some Tyr residues from the comparison of the n ear-UV CD spectra of the PI3K SH3 domain with or without a D21N mutation [O kishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affec ted Tyr residues were identified as Tyr14, and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Ph e residue with or without a D21N mutation. The H-1 and N-15 resonance assig nments of the PI3K SH3 domain and its D21N mutant revealed that significant chemical shift changes occurred to the aromatic side-chain protons of Trp5 5 and Tyr14 upon the D21N mutation, All these aromatic residues are implica ted in ligand recognition. In addition, the NMR analysis showed that the ba ckbone conformations of Lys 15-Asp23, Gly54-Trp55, Asn57-Gly58, and Gly67-P ro70 were affected by the D21N mutation. Furthermore, the 15N{H-1} nuclear Overhauser effect values of Tyr14, Glu19, and Glu20 were remarkably changed by the mutation. These results show that the D21N mutation causes structur al deformation of more than half of the ligand binding cleft of the domain and provide evidence that Asp21 plays an important role in forming a well-o rdered ligand binding cleft in cooperation with the RT loop (Lys15-Glu20).