(672d) Docking Refinement and Molecular Dynamics Studies Elucidating the Molecular Mechanism of Attractant Signaling to Dhma By E. coli Tsr (Faculty Candidate)

The study of protein-small molecule ligand interactions is a key subject in understanding protein function in biological systems. Computational modeling and molecular dynamics simulations can be coupled with experimental studies to provide an atomic-level resolution of such interactions and fill crucial gaps by obtaining information that is not accessible from experiments. Docking algorithms are widely used to understand the relationship between the structure and function of interactions within protein-small molecule ligand complexes that govern biological functions. Despite the advances made in computational chemistry, and the variety of docking frameworks to study small molecule : receptor binding, there is still significant room for improvement in obtaining highly accurate computational predictions of the structure of small-molecule : receptor complexes. Additionally, while the use of all-atom molecular dynamics (MD) simulations is increasingly important in the comparison of ligands binding to a receptor, the high resolution of these powerful techniques requires computational resources that scale both with the size of the complex under investigation and the amount of time simulated.

In this study, we show an application of an in-house docking-refinement¹ protocol to reveal a structural mechanism by which the Escherichia coli (E. coli) Tsr chemoreceptor distinguishes between the (R)- and (S) enantiomers of DHMA², metabolites of norepinephrine, a hormone that is released in high quantities in the gastrointestinal tract. We used the docking-refinement protocol, MD simulations, free energy calculations, and structural analyses to elucidate their binding and to predict which enantiomer induces a stronger signal upon binding to Tsr. The initial docking-refinement studies suggested a difference in Tsr-DHMA interaction and orientation between the two enantiomers. Starting with the binding modes derived from the docking-refinement, we used five 100 ns MD simulations per system to investigate the stability of the putative poses and their effect on Tsr. The MD simulations coupled with principal component analysis (PCA) and dynamic cross correlation (DCC) analysis predicted the piston-like motion by an α-helix of Tsr to be of higher magnitude for (R)- than (S)-DHMA due to differences in specific interactions of the ligands with residues in the binding pocket. This prediction justified the effort to experimentally separate the commercially available racemic mixture of (R)-DHMA and (S)-DHMA into the pure enantiomers, which could then be tested for their activity independently. The predicted difference in signaling response between the enantiomers was validated through tests of chemotaxis in vivo, showing that the computationally revealed difference between (R)- and (S)-DHMA binding corresponds to different behavioral responses in E. coli. Our study shows that the use of docking-refinement to generate near-optimal initial modes as initial structures for MD simulations can be used to avoid longer simulations³, with the conclusions of the simulation studies confirmed by experiments².

1. Orr AA, Jayaraman A, Tamamis P. Molecular Modeling of Chemoreceptor:Ligand Interactions. Methods Mol Biol. 2018;1729:353-372.
2. Orr AA, Yang J, Sule N, Chawla R, Hull KG, Zhu M, Romo D, Lele PP, Jayaraman A, Manson MD, Tamamis P. Molecular Mechanism for Attractant Signaling to DHMA by E. coli Tsr. Biophys J. 2020;118(2):492-504.
3. Podgorny AR, Ray JCJ. Tasting the Terroir with Tsr. Biophys J. 2020;118(2):279-280.