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
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- 作者：Nobuyuki Okishio ; Toshiyuki Tanaka ; Ryuji Fukuda ; and Masako Nagai
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：January 9, 2001
- 年：2001
- 卷：40
- 期：1
- 页码：119 - 129
- 全文大小：466K
- 年卷期：v.40,no.1(January 9, 2001)
- ISSN：1520-4995
文摘
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 
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 ligand 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 ligand binding cleft of thedomain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand bindingcleft in cooperation with the RT loop (Lys15-Glu20).
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 ligand 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 ligand binding cleft of thedomain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand bindingcleft in cooperation with the RT loop (Lys15-Glu20).