Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Biochemistry. 2010 Jun 15;49(23):4897-907. doi: 10.1021/bi1001344.
The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during gamma-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP.vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A.MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values.
肌球蛋白的活性位点包含一组高度保守的氨基酸残基，从最初的结构和动力学分析来看，这些残基在核苷酸水解和能量转导中的作用似乎显而易见，但在深入研究后，其作用变得不那么明显。其中一个残基是 Ser236（Dictyostelium discoideum 肌球蛋白 II 的编号），它被提出参与 ATP 的 γ-磷酸水解过程中的氢键转移网络，这意味着它在 ATP 水解和运动中具有关键功能。S236A 突变蛋白的水解活性和运动性仅略有下降，因此该残基似乎不是必需的。为了更好地理解 Ser236 对肌球蛋白功能的贡献，对 S236A 突变蛋白进行了结构和动力学研究。已经确定了 D. discoideum 运动结构域（S1dC）S236A 突变蛋白与镁焦磷酸盐、MgAMPPNP 和 MgADP.vanadate 的复合物结构。与野生型 S1dC 的先前结构相比，S236A.MgAMPPNP 复合物结晶在关闭状态。此外，瞬态动力学显示核苷酸释放步骤减少了 4 倍，表明突变稳定了封闭的活性位点。结构表明，一个水分子大约占据了镁焦磷酸盐和 MgAMPPNP 结构中 Ser236 缺失羟基的位置。这项研究表明，S236A 突变肌球蛋白的结构机制与野生型肌球蛋白不同，其中替代机制能够维持接近正常的瞬态动力学值。
