To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue Asp21 of thephosphatidylinositol 3-kinase (PI3K) SH3 domain, structural changes induced by the D21N mutation(Asp21
![](/images/entities/rarr.gif)
Asn) were examined by circular dichroism (CD)
and nuclear magnetic resonance (NMR)spectroscopies. In the previous study, we demonstrated that environmental alterations occurred at the sidechains of Trp55
and some Tyr residues from the comparison of the near-UV CD spectra of the PI3K SH3domain with or without a D21N mutation [Okishio, N., et al. (2000)
Biopolymers 57, 208-217]. In thiswork, the affected Tyr residues were identified as Tyr14
and Tyr73 by the CD analysis of a series ofmutants, in which every single Tyr residue was replaced by a Phe residue with or without a D21N mutation.The
1H
and 15N resonance assignments of the PI3K SH3 domain
and its D21N mutant revealed thatsignificant chemical shift changes occurred to the aromatic side-chain protons of Trp55
and Tyr14 uponthe D21N mutation. All these aromatic residues are implicated in lig
and recognition. In addition, theNMR analysis showed that the backbone conformations of Lys15-Asp23, Gly54-Trp55, Asn57-Gly58,
and Gly67-Pro70 were affected by the D21N mutation. Furthermore, the
15N{
1H} nuclear Overhausereffect values of Tyr14, Glu19,
and Glu20 were remarkably changed by the mutation. These results showthat the D21N mutation causes structural deformation of more than half of the lig
and binding cleft of thedomain
and provide evidence that Asp21 plays an important role in forming a well-ordered lig
and bindingcleft in cooperation with the RT loop (Lys15-Glu20).