School of Interdisciplinary Engineering and Sciences (SINES), National University of Sciences and Technology, Islamabad, Federal, Pakistan.
Department of Chemistry, Lancaster University, UK, Lancaster, United Kingdom, UK.
PeerJ. 2022 Mar 29;10:e12984. doi: 10.7717/peerj.12984. eCollection 2022.
Several natural mutants of the human G6PD enzyme exist and have been reported. Because the enzymatic activities of many mutants are different from that of the wildtype, the genetic polymorphism of G6PD plays an important role in the synthesis of nucleic acids via ribulose-5-phosphate and formation of reduced NADP in response to oxidative stress. G6PD mutations leading to its deficiency result in the neonatal jaundice and acute hemolytic anemia in human. Herein, we demonstrate the molecular dynamics simulations of the wildtype G6PD and its three mutants to monitor the effect of mutations on dynamics and stability of the protein. These mutants are Chatham (A335T), Nashville (R393H), Alhambra (V394L), among which R393H and V394L lie closer to binding site of structural NADP. MD analysis including RMSD, RMSF and protein secondary structure revealed that decrease in the stability of mutants is key factor for loss of their activity. The results demonstrated that mutations in the G6PD sequence resulted in altered structural stability and hence functional changes in enzymes. Also, the binding site, of structural NADP, which is far away from the catalytic site plays an important role in protein stability and folding. Mutation at this site causes changes in structural stability and hence functional deviations in enzyme structure reflecting the importance of structural NADP binding site. The calculation of binding free energy by post processing end state method of Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) has inferred that ligand binding in wildtype is favorable as compared to mutants which represent destabilised protein structure due to mutation that in turn may hinder the normal physiological function. Exploring individual components of free energy revealed that the van der Waals energy component representing non-polar/hydrophobic energy contribution act as a dominant factor in case of ligand binding. Our study also provides an insight in identifying the key inhibitory site in G6PD and its mutants which can be exploited to use them as a target for developing new inhibitors in rational drug design.
几种人类 G6PD 酶的天然突变体已经被报道。由于许多突变体的酶活性与野生型不同，G6PD 的遗传多态性在核糖-5-磷酸的合成和氧化应激下还原型 NADP 的形成中起着重要作用。导致 G6PD 缺乏的突变导致人类新生儿黄疸和急性溶血性贫血。在此，我们通过分子动力学模拟野生型 G6PD 及其三个突变体来监测突变对蛋白质动力学和稳定性的影响。这些突变体是 Chatham（A335T）、Nashville（R393H）、Alhambra（V394L），其中 R393H 和 V394L 更接近结构 NADP 的结合位点。包括 RMSD、RMSF 和蛋白质二级结构在内的 MD 分析表明，突变体稳定性的降低是其活性丧失的关键因素。结果表明，G6PD 序列中的突变导致酶结构的稳定性发生改变，从而导致功能发生变化。此外，远离催化位点的结构 NADP 结合位点在蛋白质稳定性和折叠中起着重要作用。该位点的突变导致结构稳定性发生变化，从而导致酶结构的功能偏差，反映了结构 NADP 结合位点的重要性。通过分子力学泊松-玻尔兹曼表面面积（MM-PBSA）后处理末端状态方法计算结合自由能推断，与突变体相比，野生型配体结合更有利，因为突变体代表了蛋白质结构的不稳定，这反过来可能会阻碍正常的生理功能。对自由能各个组成部分的探索表明，范德华力能量成分代表非极性/疏水能量贡献，在配体结合时起主要作用。我们的研究还提供了一种深入了解 G6PD 及其突变体关键抑制位点的方法，可以利用这些抑制位点作为开发合理药物设计中新抑制剂的靶点。
