Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase
Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase
复制标题
DOI：
10.1016/j.jmb.2005.07.042
复制
发表时间：
2005-10-07
影响因子：
5.6
通讯作者：
Cherfils, J
中科院分区：
文献类型：
--
作者：
Hible, G;Renault, L;Cherfils, J
鸟苷一磷酸激酶（GMPKs）催化GMP和dGMP磷酸化为它们的二磷酸形式，已经被表征为真核生物和原核生物中的单体酶。在这里，我们报告说，GMPK大肠杆菌（ecGMPK）组装在溶液中，并在晶体中作为几个不同的低聚物。热力学分析，ecGMPK使用差示扫描量热法表明，该酶是在二聚体和高阶低聚物之间的平衡，其相对量取决于蛋白质浓度，离子强度，和ATP的存在。分别以3.2埃、2.9埃和2.4埃分辨率解析apo、GMP和GDP结合形式的ecGMPK的晶体结构。ecGMPK在所有晶体形式中形成具有D3对称性的六聚体，其中两个核苷酸结合结构域能够经历与单体GMPK相当的闭合。2倍和3倍，接口涉及20个残基的C-末端延伸和序列，签名，分别从单体真核GMPK中缺失，解释了为什么ecGMPK形成寡聚体。这些特征在来自变形菌的GMPK中发现，其中一些是人类病原体。因此，来自这些细菌的GMPK可能形成相同的四级结构。在低ecGMPK浓度下，热力学平衡向二聚体的转移，以及亚基间相互作用部分封闭六聚体结构中的ATP结合位点的观察结果表明，二聚体可能是生理酶浓度下的活性物质。(c)2005爱思唯尔有限公司保留所有权利。
Guanosine monophosphate kinases (GMPKs), which catalyze the phosphorylation of GMP and dGMP to their diphosphate form, have been characterized as monomeric enzymes in eukaryotes and prokaryotes. Here, we report that GMPK from Escherichia coli (ecGMPK) assembles in solution and in the crystal as several different oligomers. Thermodynamic analysis, of ecGMPK using differential scanning calorimetry shows that the enzyme is in equilibrium between a dimer and higher order oligomers, whose relative amounts depend on protein concentration, ionic strength, and the presence of ATP. Crystallographic structures of ecGMPK in the apo, GMP and GDP-bound forms were solved at 3.2 angstrom, 2.9 angstrom and 2.4 angstrom resolution, respectively. ecGMPK forms a hexamer with D3 symmetry in all crystal forms, in which the two nucleotide-binding domains are able to undergo closure comparable to that of monomeric GMPKs. The 2-fold and 3-fold, interfaces involve a 20-residue C-terminal extension and a sequence, signature, respectively, that are missing from monomeric eukaryotic GMPKs, explaining why ecGMPK forms oligomers. These signatures are found in GMPKs from proteobacteria, some of which are human pathogens. GMPKs from these bacteria are thus likely to form the same quaternary structures. The shift of the thermodynamic equilibrium towards the dimer at low ecGMPK concentration together with the observation that inter-subunit interactions partially occlude the ATP-binding site in the hexameric structure suggest that the dimer may be the active species at physiological enzyme concentration. (c) 2005 Elsevier Ltd. All rights reserved.