Ohren Jeffrey F, Kundracik Melisa L, Borders Charles L, Edmiston Paul, Viola Ronald E
Department of Chemistry, The University of Toledo, Toledo, Ohio 43606, USA.
Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):381-9. doi: 10.1107/S0907444906056204. Epub 2007 Feb 21.
The structure of a transition-state analog complex of a highly soluble mutant (R134K) of rabbit muscle creatine kinase (rmCK) has been determined to 1.65 A resolution in order to elucidate the structural changes that are required to support and regulate catalysis. Significant structural asymmetry is seen within the functional homodimer of rmCK, with one monomer found in a closed conformation with the active site occupied by the transition-state analog components creatine, MgADP and nitrate. The other monomer has the two loops that control access to the active site in an open conformation and only MgADP is bound. The N-terminal region of each monomer makes a substantial contribution to the dimer interface; however, the conformation of this region is dramatically different in each subunit. Based on this structural evidence, two mutational modifications of rmCK were conducted in order to better understand the role of the amino-terminus in controlling creatine kinase activity. The deletion of the first 15 residues of rmCK and a single point mutant (P20G) both disrupt subunit cohesion, causing the dissociation of the functional homodimer into monomers with reduced catalytic activity. This study provides support for a structural role for the amino-terminus in subunit association and a mechanistic role in active-site communication and catalytic regulation.
为了阐明支持和调节催化作用所需的结构变化，已确定兔肌肉肌酸激酶（rmCK）的高溶解性突变体（R134K）的过渡态类似物复合物的结构，分辨率达到1.65 Å。在rmCK的功能性同型二聚体内观察到明显的结构不对称性，其中一个单体处于封闭构象，活性位点被过渡态类似物成分肌酸、MgADP和硝酸盐占据。另一个单体的两个控制进入活性位点的环处于开放构象，仅结合了MgADP。每个单体的N端区域对二聚体界面有很大贡献；然而，该区域在每个亚基中的构象差异很大。基于这一结构证据，对rmCK进行了两种突变修饰，以便更好地了解氨基末端在控制肌酸激酶活性中的作用。rmCK前15个残基的缺失和单点突变体（P20G）均破坏了亚基凝聚力，导致功能性同型二聚体解离为催化活性降低的单体。这项研究为氨基末端在亚基缔合中的结构作用以及在活性位点通讯和催化调节中的机制作用提供了支持。
