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A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP
Alexander Krah1, Bas van der Hoeven2, Luuk Mestrom2
1Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan; Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Str., #07-01 Matrix, 138671, Singapore.
Biochimica Et Biophysica Acta. General Subjects
|October 18, 2020
Summary
Molecular dynamics (MD) simulations revealed that a specific residue outside the ATP binding site significantly impacts ATP binding affinity in bacterial ATP synthase ε subunits. This finding advances protein design and understanding of enzyme function.
Area of Science:
- Biochemistry
- Structural Biology
- Computational Biology
Background:
- Enzyme function and ligand binding prediction are complex and costly.
- Proteins are functionally dynamic, not static as crystallography suggests.
- Molecular dynamics (MD) can probe protein movement and predict ligand binding.
Purpose of the Study:
- Investigate the ~500-fold difference in ATP affinity between Bacillus subtilis and Bacillus PS3 ε subunits.
- Determine the influence of residues outside the ATP-binding site on affinity.
- Validate MD simulation predictions experimentally.
Main Methods:
- Modeled Bacillus subtilis ε subunit structure using Bacillus PS3 ε subunit structure.
- Employed MD simulations to predict the effect of external residues on ATP binding.
- Created point mutants and performed ATP binding studies to verify predictions.
Main Results:
- MD simulations identified residue E102 in B. subtilis ε subunit as crucial for ATP binding affinity.
- Mutating E102 to alanine or arginine increased ATP binding by ~10-fold and ~54-fold, respectively.
- Experimental results confirmed MD predictions regarding E102's significant influence.
Conclusions:
- MD simulations can predict how "second shell" residues affect substrate binding affinity.
- Explains the varying ATP binding affinities in seemingly identical ε subunits.
- Highlights the potential of MD for protein design and functional studies.
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