Gráczer Éva, Merli Angelo, Singh Rajesh Kumar, Karuppasamy Manikandan, Závodszky Péter, Weiss Manfred S, Vas Mária
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, PO Box 7, H1518 Budapest, Hungary.
Mol Biosyst. 2011 May;7(5):1646-59. doi: 10.1039/c0mb00346h. Epub 2011 Mar 8.
The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 Å resolution, in complexes with both Mn(2+) and 3-isopropylmalate (IPM), as well as with both Mn(2+) and NADH, were determined at resolutions ranging from 2.0 to 2.5 Å. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn(2+) and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and βF as well as hinge 2 between αh and βE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn(2+) and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements βE, βF, βG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 Å upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions.
基于嗜热栖热菌同二聚体异丙基苹果酸脱氢酶（IPMDH）的X射线结构的成对比较及其详细的分子图形分析，在原子水平上描述了与催化循环相关的结构域闭合。测定了无底物的脱辅基形式、与二价金属离子形成复合物至1.8 Å分辨率、与Mn(2+)和3-异丙基苹果酸（IPM）形成复合物以及与Mn(2+)和NADH形成复合物的结构，分辨率范围为2.0至2.5 Å。单晶显微分光光度测量表明，在存在Mn(2+)和IPM的情况下生长的晶体中存在功能上有效的蛋白质构象。各种复合物的结构比较清楚地揭示了每个亚基内两个结构域的相对运动，并允许识别结构域间区域的两个铰链：αd和βF之间的铰链1以及αh和βE之间的铰链2。对保守氨基酸侧链的原子接触的详细分析表明了这些分子铰链在底物作用下的可能作用机制。蛋白质与Mn(2+)和IPM的相互作用主要负责结构域闭合：底物IPM结合到结构域间区域的裂隙中时，会诱导二级结构元件βE、βF、βG、αd和αh的相对运动。构象变化的另一个特殊特征是携带亚基相互作用臂之前的氨基酸Tyr139的环的运动。IPM结合后，酪氨酸环旋转并移动至少5 Å。从而，在IPM的埋藏异丙基上方形成了新的疏水相互作用。然后仅通过亚基相互作用完成结构域闭合：一个亚基的环插入另一个亚基的结构域间腔中，扩展了具有疏水相互作用的区域，提供了结构域间和亚基间相互作用协同作用的一个例子。
