Borkotoky Subhomoi, Murali Ayaluru
Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Puducherry, 605014, India.
BMC Struct Biol. 2017 May 25;17(1):7. doi: 10.1186/s12900-017-0077-9.
T7 lysozyme (T7L), also known as N-acetylmuramoyl-L-alanine amidase, is a T7 bacteriophage gene product. It involves two functions: It can cut amide bonds in the bacterial cell wall and interacts with T7 RNA polymerase (T7RNAP) as a part of transcription inhibition. In this study, with the help of molecular dynamics (MD) calculations and computational interaction studies, we investigated the effect of varying pH conditions on conformational flexibilities of T7L and their influence on T7RNAP -T7L interactions.
From the MD studies of the T7L at three different pH strengths viz. 5, neutral and 7.9 it was observed that T7L structure at pH 5 exhibited less stable nature with more residue level fluctuations, decrease of secondary structural elements and less compactness as compared to its counterparts: neutral pH and pH 7.9. The T-pad analysis of the MD trajectories identified local fluctuations in few residues that influenced the conformational differences in three pH strengths. From the docking of the minimum energy representative structures of T7L at different pH strengths (obtained from the free energy landscape analysis) with T7RNAP structures at same pH strengths, we saw strong interaction patterns at pH 7.9 and pH 5. The MD analysis of these complexes also confirmed the observations of docking study. From the combined in silico studies, it was observed that there are conformational changes in N-terminal and near helix 1 of T7L at different pH strengths, which are involved in the T7RNAP interaction, thereby varying the interaction pattern.
Since T7L has been used for developing novel therapeutics and T7RNAP one of the most biologically useful protein in both in-vitro and in vivo experiments, this in silico study of pH dependent conformational differences in T7L and the differential interaction with T7RNAP at different pH can provide a significant insight into the structural investigations on T7L and T7RNAP in varying pH environments.
T7溶菌酶（T7L），也被称为N - 乙酰胞壁酰 - L - 丙氨酸酰胺酶，是T7噬菌体的基因产物。它具有两种功能：能够切断细菌细胞壁中的酰胺键，并作为转录抑制的一部分与T7 RNA聚合酶（T7RNAP）相互作用。在本研究中，借助分子动力学（MD）计算和计算相互作用研究，我们研究了不同pH条件对T7L构象灵活性的影响及其对T7RNAP - T7L相互作用的影响。
通过对T7L在三种不同pH强度（即5、中性和7.9）下的MD研究观察到，与中性pH和pH 7.9相比，pH 5时T7L结构表现出较低的稳定性，残基水平波动更多，二级结构元件减少且紧凑性降低。对MD轨迹的T - 垫分析确定了少数残基的局部波动，这些波动影响了三种pH强度下的构象差异。通过将不同pH强度下T7L的最低能量代表性结构（从自由能景观分析中获得）与相同pH强度下的T7RNAP结构进行对接，我们在pH 7.9和pH 5时看到了强烈的相互作用模式。这些复合物的MD分析也证实了对接研究的观察结果。从综合的计算机模拟研究中观察到，在不同pH强度下，T7L的N端和靠近螺旋1处存在构象变化，这些变化参与了T7RNAP相互作用，从而改变了相互作用模式。
由于T7L已被用于开发新型疗法，且T7RNAP是体外和体内实验中最具生物学用途的蛋白质之一，因此这项关于T7L中pH依赖性构象差异以及在不同pH下与T7RNAP的差异相互作用的计算机模拟研究，能够为在不同pH环境下对T7L和T7RNAP的结构研究提供重要的见解。
