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A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-10-15 , DOI: 10.1016/j.bbagen.2020.129766
Alexander Krah , Bas van der Hoeven , Luuk Mestrom , Fabio Tonin , Kirsten C.C. Knobel , Peter J. Bond , Duncan G.G. McMillan

Background

Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site.

Methods

We first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed.

Results

MD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity.

Conclusions

These findings reveal how MD can predict how changes in the “second shell” residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities.

General significance

This study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.



中文翻译:

第二个壳残基以与ATP的亲和力完全不同的方式调节保守的ATP结合位点

背景

配体结合的预测和酶新功能的设计是一个耗时且昂贵的过程。晶体学给人的印象是蛋白质呈固定形状,而酶在功能上是动态的。分子动力学提供了在预测配体结合的同时探测蛋白质运动的可能性。因此,我们选择细菌F 1 F o ATP合酶ε亚基来揭示为什么枯草芽孢杆菌芽孢杆菌PS3的ε亚基的ATP亲和力为何相异〜500倍,尽管在ATP结合位点共享相同的序列。

方法

我们首先使用芽孢杆菌PS3ε亚基结构来模拟枯草芽孢杆菌ε亚基结构,并以此来探索分子动力学(MD)模拟的效用,以预测ATP结合位点以外的残基的影响。为了验证MD预测,进行了点突变,并进行了ATP结合研究。

结果

MD模拟预测,枯草芽孢杆菌ε亚基在ATP结合位点之外的E102影响ATP结合亲和力。对丙氨酸或精氨酸进行工程改造的E102分别显示出ATP结合增加了约10倍或约54倍,从而证实了MD预测E102会严重影响ATP结合亲和力。

结论

这些发现揭示了MD如何预测底物结合位点周围“第二壳”残基的变化如何影响简单蛋白质结构中的亲和力。我们的结果揭示了为什么不同ATP合酶中看似相同的ε亚基具有根本不同的ATP结合亲和力。

一般意义

这项研究可能会导致分子动力学作为蛋白质设计工具以及蛋白质设计和功能探索的更大用途。

更新日期:2020-10-30
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