Abstract：
MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded \"opened\" and liganded \"closed\" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo \"opened\" and an artificial generated apo \"closed\" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.
摘要：
MshA是一种GT-B糖基转移酶，催化真菌硫醇生物合成的第一步。虽然许多GT-B酶经历开放到封闭的转变，MshA是独一无二的，因为它的97°旋转超出了通常的10-25°范围。对配体结合和未结合状态的MshA进行了分子动力学（MD）模拟，以研究配体结合对局部蛋白质动力学及其构象自由能景观的影响。模拟表明，酶样本的无配体“开放”和配体“封闭”形式都具有很大程度的二面角和域间距离，重叠种群相对较低。使用apo“开放”和人工生成的apo“封闭”结构的副本交换MD计算自由能表面，揭示了采样的几何形状中的重叠，允许在不存在配体的情况下计算2kcal/mol的开放到封闭过渡的势垒。完全结合的MshA的MD模拟显示二面角的采样较小。局部蛋白质波动变化表明，尽管与UDP-GlcNAc的相互作用几乎没有变化，但UDP-GlcNAc结合激活了1-1-肌醇-1-磷酸（I1P）结合位点中的环运动。圆二色性,本征荧光光谱，和诱变研究用于确认MshA中配体诱导的结构变化。结果支持提出的机制，其中UDP-GlcNAc与MshA的C端结构域刚性相互作用结合，并激活N端结构域中的柔性环以结合和定位I1P。该模型可用于未来的基于结构的药物开发中的mycohiol生物合成途径的抑制剂。
