Mercier Evan, Girodat Dylan, Wieden Hans-Joachim
Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.
Sci Rep. 2015 Jan 8;5:7677. doi: 10.1038/srep07677.
The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu · GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C. We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.
磷酸结合环（P环）是在单核苷酸结合蛋白中发现的保守序列基序。关于该区域的结构动力学及其对观察到的核苷酸结合特性的贡献知之甚少。理解其潜在的设计原则对于生物分子工程应用具有重要意义。我们利用体外快速动力学测量和计算机分子动力学（MD）模拟来研究普遍保守的延伸因子（EF）Tu中GTP结合特性与P环结构动力学之间的关系。对野生型EF-Tu以及P环内或其邻近位置有替换的变体进行分析，揭示了P环灵活性与GTP解离活化熵之间的相关性。在引导分子动力学模拟中，相同的变体在力诱导的EF-Tu·GTP解离过程中，P环的两个N端氨基酸表现出更大的主链灵活性。甘氨酸18和组氨酸19通过与相邻的螺旋C相互作用参与稳定P环主链。我们提出这些P环/螺旋C相互作用作为一个保守的P环锚定模块发挥作用，并确定在几种GTP酶和ATP酶中存在P环锚，表明它们在进化上的保守性。
