Perona J J, Martin A M
Department of Chemistry, University of California at Santa Barbara 93106-9510, USA.
J Mol Biol. 1997 Oct 17;273(1):207-25. doi: 10.1006/jmbi.1997.1315.
The structures of wild-type and mutant forms of the unliganded EcoRV endonuclease dimer have been determined at 2.4 A resolution in a new crystal lattice. Comparison of these structures with that of the free enzyme determined with different packing constraints shows that the conformations of the domain interfaces are not conserved between crystal forms. The unliganded enzyme and the enzyme-DNA complex delineate two distinct quaternary states separated by a 25 degrees intersubunit rotation, but considerable conformational heterogeneity, of the order of 10 degrees domain rotations, exists within each of these states. Comparison of the free enzyme structure between the two crystal forms further reveals that the C-terminal 28 amino acid residues are disordered and undergo an extensive local folding transition upon DNA binding. Introduction of the mutation T93A at the DNA-binding cleft causes large-scale effects on the protein conformation. Structural changes in the mutated unliganded enzyme propagate some 20 to 25 A to the dimerization interface and lead to a rearrangement of monomer subunits. Comparative analysis of these structures, a new structure of the enzyme cocrystallized with DNA and calcium ions, and previously determined cocrystal structures suggests important roles for a number of amino acid residues in facilitating the intersubunit motions and local folding transitions. In particular, the T93A structure reveals a pathway through the protein, by which DNA-binding may cause the domain movements required for proper alignment of catalytic groups. The key active-site residue Glu45 is located on a flexible helix inside this pathway, and this provides a direct means by which essential catalytic functions are coupled to the protein conformational change. It appears that indirect perturbation of the Glu45 conformation via an altered quaternary structure may be a contributing factor to the decreased catalytic efficiency of T93A, and this mechanism may also explain the diminished activities of other active site variants of EcoRV.
在一个新的晶格中，已在2.4埃分辨率下测定了未结合配体的EcoRV核酸内切酶二聚体的野生型和突变体形式的结构。将这些结构与在不同堆积限制条件下测定的游离酶的结构进行比较，结果表明，不同晶体形式之间结构域界面的构象并不保守。未结合配体的酶和酶-DNA复合物描绘了两种不同的四级状态，它们之间存在25度的亚基间旋转，但在每种状态下都存在相当大的构象异质性，其量级为10度的结构域旋转。对两种晶体形式的游离酶结构进行比较进一步发现，C末端的28个氨基酸残基是无序的，并且在DNA结合时会经历广泛的局部折叠转变。在DNA结合裂隙处引入T93A突变会对蛋白质构象产生大规模影响。突变的未结合配体的酶中的结构变化会传播约20至25埃到二聚化界面，并导致单体亚基的重排。对这些结构、与DNA和钙离子共结晶的酶的新结构以及先前测定的共晶体结构进行比较分析，表明许多氨基酸残基在促进亚基间运动和局部折叠转变中起重要作用。特别是，T93A结构揭示了一条贯穿蛋白质的途径，通过该途径DNA结合可能导致催化基团正确排列所需的结构域运动。关键的活性位点残基Glu45位于该途径内的一个柔性螺旋上，这提供了一种直接方式，通过它基本的催化功能与蛋白质构象变化相耦合。看来，通过改变的四级结构对Glu45构象的间接扰动可能是T93A催化效率降低的一个促成因素，并且这种机制也可能解释EcoRV其他活性位点变体活性降低的原因。
