Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Biochemistry. 2011 Apr 12;50(14):2860-9. doi: 10.1021/bi200039u. Epub 2011 Mar 16.
Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.
蛋白激酶 C 相关激酶 (PRKs) 是丝氨酸/苏氨酸激酶，属于蛋白激酶 C 超家族成员，可通过与 Rho 家族的小 G 蛋白结合来激活，这种结合通过一个 Rho 结合基序（称为 HR1 结构域）实现。PRKs 在其 N 端包含三个串联的 HR1 结构域。PRK1 的 HR1a 结构域与 RhoA 形成的复合物结构 [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] 确定了 G 蛋白与 HR1a 结构域之间的两个潜在接触界面。在这项工作中，我们使用丙氨酸扫描诱变方法来确定这两种蛋白质在溶液中相互作用时是否使用了这两个接触点，以及 PRK1 的第二个 HR1 结构域 HR1b 是否在与 RhoA 结合中发挥作用。突变分析确定了一个接触点与 RhoA 和 HR1a 在溶液中的结合相关，而 HR1b 结构域未发现与 RhoA 结合。还研究了 HR1a 和 HR1b 结构域的折叠状态和热稳定性。发现 HR1b 比 HR1a 更热稳定，据推测，这两个结构域的生物物理性质差异决定了它们与小 G 蛋白的相互作用。
