Duée E, Olivier-Deyris L, Fanchon E, Corbier C, Branlant G, Dideberg O
Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale Jean-Pierre EBEL, Grenoble, France.
J Mol Biol. 1996 Apr 12;257(4):814-38. doi: 10.1006/jmbi.1996.0204.
The crystal structure of wild-type and N313T mutant glyceraldehyde 3-phosphate dehydrogenases from Escherichia coli was determined in the presence of NAD at 1.8 angstrom and 2.17 angstrom, respectively. The structure of the monomer and of the tetramer are similar to those observed for other GAPDHs. An exhaustive analysis of the hydrophobic clusters and the hydrogen bond networks explain the high degree of sequence conservation in GAPDHs. The structural effect of the N313T mutation is a change in the (phi,psi) angles of nearby residues Asn236 and Val237, while the structure around the mutated residue remains unchanged. A detailed comparison of the wild-type and N313T mutant E. coli GAPDH with the apo and holo forms of Bacillus stearothermophilus GAPDH is carried out in relation to the apo --> holo transition. An unbiased set of about 60 residues, whose C(alpha) atoms remain in the same relative position in the different forms of the tetramer, is defined as the tetramer "core" which acts as a fixed scaffold around which structural rearrangements occur during the apo --> holo transition. This core essentially includes beta-strands from the beta-sheets forming the O-P and Q-R interfaces, in particular strand beta1 which bears catalytic residue His176. During the apo --> holo transition, dimer O-P rotates around the molecular P-axis by about +1 degrees, and dimer O-R by about -1 degrees. Further rotations of the NAD binding domain relative to the catalytic domain are discussed in relation to the molecular symmetry. The possible effect on NAD binding cooperativity of mutations around the tetramer core is exemplified by residue 252. The presence of a conserved hydrophilic patch embedded in the hydrophobic O-P interface is highlighted. A mechanism for substrate binding, different from those currently proposed, is described where the hydroxyl group of the substrate C(2) atom is hydrogen bonded to Cys149N.
分别在存在NAD的情况下，测定了来自大肠杆菌的野生型和N313T突变型甘油醛-3-磷酸脱氢酶的晶体结构，分辨率分别为1.8埃和2.17埃。单体和四聚体的结构与其他甘油醛-3-磷酸脱氢酶（GAPDHs）的结构相似。对疏水簇和氢键网络的详尽分析解释了GAPDHs中高度的序列保守性。N313T突变的结构效应是附近残基Asn236和Val237的（φ，ψ）角发生变化，而突变残基周围的结构保持不变。针对apo→全酶转变，对野生型和N313T突变型大肠杆菌GAPDH与嗜热栖热芽孢杆菌GAPDH的脱辅基和全酶形式进行了详细比较。一组约60个残基，其Cα原子在四聚体的不同形式中保持相同的相对位置，被定义为四聚体“核心”，它作为一个固定的支架，在apo→全酶转变过程中围绕其发生结构重排。这个核心主要包括来自形成O-P和Q-R界面的β-折叠的β-链，特别是带有催化残基His176的β1链。在apo→全酶转变过程中，二聚体O-P围绕分子P轴旋转约+1度，二聚体O-R旋转约-1度。还讨论了NAD结合结构域相对于催化结构域的进一步旋转与分子对称性的关系。以残基252为例说明了四聚体核心周围突变对NAD结合协同性的可能影响。突出了疏水O-P界面中嵌入的保守亲水斑块的存在。描述了一种不同于目前提出的底物结合机制，其中底物C(2)原子的羟基与Cys149N形成氢键。
