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Abstract The catalytic activity of human glutathione S‐transferase A1‐1 (hGSTA1‐1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C‐terminal helix α 9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand‐free state of the hGSTA1‐1 homodimer has not been resolved. Here, we used a combination of EPR distance measurements and weighted ensemble simulations to characterize the conformational ensemble of the ligand‐free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand‐free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α 9 helices upon ligand binding. Weighted ensemble simulations generated unbiased pathways for the seconds‐timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand‐free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1‐1, which involve the mutually exclusive docking of α 9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1‐1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with weighted ensemble rare‐events sampling strategy to gain mechanistic information on protein function at the atomic level. This article is protected by copyright. All rights reserved.
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Kinetics and free energy of ligand dissociation using weighted ensemble milestoning.
We consider the recently developed weighted ensemble milestoning (WEM) scheme [D. Ray and I. Andricioaei, J. Chem. Phys. 152, 234114 (2020)] and test its capability of simulating ligand-receptor dissociation dynamics. We performed WEM simulations on the following host-guest systems: Na+/Cl- ion pair and 4-hydroxy-2-butanone ligand with FK506 binding protein. As a proof of principle, we show that the WEM formalism reproduces the Na+/Cl- ion pair dissociation timescale and the free energy profile obtained from long conventional MD simulation. To increase the accuracy of WEM calculations applied to kinetics and thermodynamics in protein-ligand binding, we introduced a modified WEM scheme called weighted ensemble milestoning with restraint release (WEM-RR), which can increase the number of starting points per milestone without adding additional computational cost. WEM-RR calculations obtained a ligand residence time and binding free energy in agreement with experimental and previous computational results. Moreover, using the milestoning framework, the binding time and rate constants, dissociation constants, and committor probabilities could also be calculated at a low computational cost. We also present an analytical approach for estimating the association rate constant (kon) when binding is primarily diffusion driven. We show that the WEM method can efficiently calculate multiple experimental observables describing ligand-receptor binding/unbinding and is a promising candidate for computer-aided inhibitor design.
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- Award ID(s):
- 2028443
- PAR ID:
- 10227750
- Date Published:
- Journal Name:
- Chemical physics
- Volume:
- 153
- Issue:
- 15
- ISSN:
- 0301-0104
- Page Range / eLocation ID:
- 154117
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1063/5.0021953
