Dynamical-nonequilibrium alanine scanning for identifying allosteric positions in proteins
Balazs Balega1, Michael Beer1, Dan T S Pike1
1Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, UK.
Biophysical Journal
|July 18, 2026
Summary
Predicting protein allostery is difficult. A new dynamical-nonequilibrium molecular dynamics (D-NEMD) method uses alanine substitutions to rapidly identify key distal sites influencing enzyme function, aiding drug resistance studies.
Area of Science:
- Biochemistry
- Computational Biology
- Structural Biology
Background:
- Allosteric regulation is crucial in proteins, but predicting the effects of distant mutations is challenging due to complex dynamical networks.
- Experimental methods for identifying functionally important distal sites are often low-throughput and labor-intensive.
- Equilibrium molecular dynamics (MD) can identify functional sites but requires extensive simulation time for each mutant.
Purpose of the Study:
- To introduce and validate a novel dynamical-nonequilibrium MD (D-NEMD) protocol for efficiently identifying distal positions that allosterically influence enzyme function.
- To demonstrate the utility of D-NEMD in distinguishing functional from nonfunctional distal sites by analyzing the propagation of perturbations.
- To apply D-NEMD to the clinically relevant KPC-2 β-lactamase to identify sites relevant to antibiotic resistance.
Main Methods:
- Development of a D-NEMD protocol employing targeted alanine substitutions as perturbations.
- Application of D-NEMD to the KPC-2 β-lactamase system to analyze the propagation of structural responses from mutated sites.
- Comparison of D-NEMD results with known experimental data on KPC-2 mutations and their impact on enzyme function and resistance phenotypes.
Main Results:
- D-NEMD successfully distinguished functional distal sites from nonfunctional ones by assessing the long-range propagation of alanine substitution effects.
- Mutations at KPC-2 positions 179, 164, and 220 showed structured, long-range responses reaching the active site, consistent with known functional roles.
- Substitution at position 276, known to have minimal kinetic impact, resulted in only local displacements, validating the method's specificity.
Conclusions:
- D-NEMD offers a fast, generalizable, and predictive approach for identifying allosterically connected distal protein positions.
- The protocol complements equilibrium MD simulations and provides a tractable method for prioritizing candidate sites for further investigation.
- This approach can accelerate mechanistic studies and mutational scanning efforts for understanding enzyme function and drug resistance.
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