A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP

Alexander Krah, Bas van der Hoeven, Luuk Mestrom, Fabio Tonin, Kirsten C.C. Knobel, Peter J. Bond, Duncan G.G. McMillan

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Abstract

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.

Original languageEnglish
Article number129766
JournalBiochimica et Biophysica Acta - General Subjects
Volume1865
Issue number1
DOIs
Publication statusPublished - 2021

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