Cronet P, Bellsolell L, Sander C, Coll M, Serrano L
European Molecular Biology Laboratory, Heidelberg, Germany.
J Mol Biol. 1995 Jun 9;249(3):654-64. doi: 10.1006/jmbi.1995.0326.
Amongst the superfamily of nucleotide binding proteins, the classical mononucleotide binding fold (CMBF), is the one that has been best characterized structurally. The common denominator of all the members is the triphosphate/Mg2+ binding site, whose signature has been recognized as two structurally conserved stretches of residues: the Kinase 1 and 2 motifs that participate in triphosphate and Mg2+ binding, respectively. The Kinase 1 motif is borne by a loop (the P-loop), whose structure is conserved throughout the whole CMBF family. The low sequence similarity between the different members raises questions about which interactions are responsible for the active structure of the P-loop. What are the minimal requirements for the active structure of the P-loop? Why is the P-loop structure conserved despite the diverse environments in which it is found? To address this question, we have engineered the Kinase 1 and 2 motifs into a protein that has the CMBF and no nucleotide binding activity, the chemotactic protein from Escherichia coli, CheY. The mutant does not exhibit any triphosphate/Mg2+ binding activity. The crystal structure of the mutant reveals that the engineered P-loop is in a different conformation than that found in the CMBF. This demonstrates that the native structure of the P-loop requires external interactions with the rest of the protein. On the basis of an analysis of the conserved tertiary contacts of the P-loop in the mononucleotide binding superfamily, we propose a set of residues that could play an important role in the acquisition of the active structure of the P-loop.
在核苷酸结合蛋白超家族中，经典单核苷酸结合折叠（CMBF）在结构上的特征最为明确。所有成员的共同特征是三磷酸/Mg2+结合位点，其特征被认为是两个结构保守的残基片段：分别参与三磷酸和Mg2+结合的激酶1和2基序。激酶1基序由一个环（P环）携带，其结构在整个CMBF家族中保守。不同成员之间较低的序列相似性引发了关于哪些相互作用负责P环活性结构的问题。P环活性结构的最低要求是什么？尽管P环存在于不同环境中，但其结构为何保守？为了解决这个问题，我们将激酶1和2基序设计到一种具有CMBF但无核苷酸结合活性的蛋白质中，即来自大肠杆菌的趋化蛋白CheY。该突变体不表现出任何三磷酸/Mg2+结合活性。突变体的晶体结构表明，设计的P环与CMBF中的P环构象不同。这表明P环的天然结构需要与蛋白质的其余部分进行外部相互作用。基于对单核苷酸结合超家族中P环保守三级接触的分析，我们提出了一组可能在P环活性结构形成中起重要作用的残基。
