Ma Hailun, Lewis David, Xu Cheng, Inesi Giuseppe, Toyoshima Chikashi
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Biochemistry. 2005 Jun 7;44(22):8090-100. doi: 10.1021/bi050332m.
Twenty five amino acids within the "N", "P", and "A" domains of the Ca(2+) ATPase (SERCA1) headpiece were subjected to site directed mutagenesis, taking advantage of a high yield expression system. Functional and conformational effects of mutations were interpreted systematically in the light of the high resolution WT structure, defining direct involvement in catalysis as well as in stabilization of various positions acquired by each domain upon substrate binding and utilization. Amino acids involved in binding of ATP (such as Phe487 and Arg560 in the N domain) or phosphate (such as Asp351, Thr625, Lys684, and Thr353 in the P domain) were characterized with respect to their binding mechanism. Further identified were "positional" roles of several amino acids that stabilize neighboring residues for optimal binding of substrate or Mg(2+), or interface between headpiece domains as they change their relative positions in the course of the catalytic cycle. These include cross-linking of the "N" and "P" domains (e.g., Arg560/Asp627 salt bridge to stabilize domain approximation by ATP binding), and stabilization of the "A", "N", and activated "P" domains in arrangements differing from the ground E2 state and driven by catalytic events. This stabilization is produced through hydrogen bonds at domain interfaces, which vary depending on the intermediate state (e.g., Glu486/T171 in E1P and E2P, as opposed to Glu486/H190 in E2). We demonstrate that specific arrangements of the headpiece domains shown in crystal structures are, in fact, required to trigger displacement of transmembrane segments during the enzyme cycle in solution, allowing long range linkage of catalytic and Ca(2+) binding functions.
利用高产表达系统，对钙（2+）ATP酶（SERCA1）头部“N”“P”和“A”结构域内的25个氨基酸进行了定点诱变。根据高分辨率野生型结构，系统地解释了突变的功能和构象效应，确定了各个结构域在底物结合和利用时所获得的不同位置直接参与催化以及稳定作用。对参与ATP结合（如N结构域中的苯丙氨酸487和精氨酸560）或磷酸结合（如P结构域中的天冬氨酸351、苏氨酸625、赖氨酸684和苏氨酸353）的氨基酸的结合机制进行了表征。还进一步确定了几个氨基酸的“位置”作用，这些氨基酸稳定相邻残基以实现底物或镁（2+）的最佳结合，或者在催化循环过程中头部结构域改变其相对位置时稳定头部结构域之间的界面。这些作用包括“N”和“P”结构域的交联（例如，精氨酸560/天冬氨酸627盐桥通过ATP结合来稳定结构域接近），以及“A”“N”和活化的“P”结构域在不同于基态E2状态且由催化事件驱动的排列中的稳定。这种稳定是通过结构域界面处的氢键产生的，氢键根据中间状态而变化（例如，E1P和E2P中的谷氨酸486/苏氨酸171，与E2中的谷氨酸486/组氨酸190相反）。我们证明，晶体结构中所示的头部结构域的特定排列实际上是在溶液中的酶循环过程中触发跨膜片段位移所必需的，从而实现催化和钙（2+）结合功能的远程连接。
