Tessari M, Gentile L N, Taylor S J, Shalloway D I, Nicholson L K, Vuister G W
Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Biochemistry. 1997 Nov 25;36(47):14561-71. doi: 10.1021/bi9712044.
The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.
本文展示了对pp60 c-Src的Src同源结构域2和3组合（SH3-SH2）进行异核核磁共振研究的结果。通过异核三共振实验结合15N分离核Overhauser效应光谱（NOESY）数据获得了共振归属。本文提出了一种改进灵敏度的三维13CO-15N-1H光谱相关实验[（HACA）CO（CA）-NH]，该实验提供了额外的序列信息并解决了一些模糊性问题。化学位移以及序列和中程NOE交叉峰表明，多肽的SH3和SH2部分的结构与分离的SH3和SH2结构域的结构非常相似。高亲和力磷酸肽EPQpYEEIPIYL的结合在SH2结构域的几个位置引起了较大的化学位移变化。与肽结合到SH2结构域的已知结果进行比较表明，显示出最大效应的残基都参与了肽结合或在结合时经历了显著的构象变化。然而，在SH3结构域和连接接头区域内的残基上观察到了1H和15N化学位移的细微变化，表明可能存在跨结构域通讯。
