Szilágyi A N, Ghosh M, Garman E, Vas M
Institute of Enzymology Biological Research Center, Hungarian Academy of Sciences, Budapest, H-1518, Hungary.
J Mol Biol. 2001 Feb 23;306(3):499-511. doi: 10.1006/jmbi.2000.4294.
3-phosphoglycerate kinase (PGK) is a typical kinase with two structural domains. The domains each bind one of the two substrates, 3-phosphoglycerate (3-PG) and MgATP. For the phospho-transfer reaction to take place the substrates must be brought closer by a hinge-bending domain closure. Open and closed structures of the enzyme with different relative domain positions have been determined from different species, but a comprehensive description of this conformational transition is yet to be attained. Crystals of pig muscle PGK in complex with MgADP and 3-phosphoglycerate were grown under the conditions which have previously resulted in crystals of the closed, catalytically competent conformation of Trypanosoma brucei PGK. The X-ray structure of the pig muscle ternary complex was determined at 1.8 A and the model was refined to R=20.8% and Rfree=24.1%. Contrary to expectation, however, it represents an essentially open conformation compared to that of T. brucei PGK. In addition, the beta-phosphate group of ADP is mobile in the new structure, in contrast to its well-defined position in T. brucei PGK. An extensive comparison of the ternary complexes from these remote species has been carried out in order to establish general differences between the two conformations and is reported here. A second pair of the open and closed structures was also compared. These analyses have made it possible to define several characteristic changes which accompany the structural transition, in addition to those identified previously: (1) the operation of a hinge at beta-strand L in the inter-domain region which greatly affects the relative domain positions; (2) the rearrangement and movement of helix 8, regulated through the interactions with the nucleotide phosphate; and (3) the existence of another hinge between helix 14 and the rest of the C-terminal part of the chain, which allows fine adjustment of the N-domain position. The main hinge at beta-strand L acts in concert with the C-terminal hinge at helix 7 described previously. Simultaneous interactions of the nucleotide phosphate groups with the loop that precedes helix 8, beta-strand J and the N terminus of helix 13 are required for propagation of the nucleotide effect towards the beta-strand L molecular hinge. A detailed description of the role of nucleotide binding in the hinge operation is presented.
3-磷酸甘油酸激酶（PGK）是一种典型的激酶，具有两个结构域。这两个结构域分别结合两种底物之一，即3-磷酸甘油酸（3-PG）和MgATP。为了发生磷酸转移反应，底物必须通过铰链弯曲使结构域闭合而靠近。已经从不同物种中确定了具有不同相对结构域位置的该酶的开放和闭合结构，但尚未获得对这种构象转变的全面描述。在先前已得到布氏锥虫PGK闭合的、具有催化活性构象晶体的条件下，培养出了与MgADP和3-磷酸甘油酸结合的猪肌肉PGK晶体。猪肌肉三元复合物的X射线结构在1.8埃分辨率下测定，模型精修至R = 20.8%，Rfree = 24.1%。然而，与预期相反，与布氏锥虫PGK相比，它代表一种基本开放的构象。此外，在新结构中ADP的β-磷酸基团是可移动的，这与它在布氏锥虫PGK中明确的位置形成对比。为了确定两种构象之间的一般差异，对来自这些远缘物种的三元复合物进行了广泛比较，并在此报告。还比较了另一对开放和闭合结构。这些分析使得除了先前确定的那些变化之外，还能够定义伴随结构转变的几个特征性变化：（1）结构域间区域中β链L处的铰链作用，这极大地影响了相对结构域位置；（2）通过与核苷酸磷酸基团的相互作用调节的螺旋8的重排和移动；（3）螺旋14与链的C末端其余部分之间存在另一个铰链，这允许对N结构域位置进行微调。β链L处的主要铰链与先前描述的螺旋7处的C末端铰链协同作用。核苷酸效应向β链L分子铰链的传播需要核苷酸磷酸基团与螺旋8之前的环、β链J和螺旋13的N末端同时相互作用。本文给出了核苷酸结合在铰链作用中作用的详细描述。
