RT Journal Article
SR Electronic
T1 Molecular-Dynamics Simulation Methods for Macromolecular Crystallography
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.04.04.486986
DO 10.1101/2022.04.04.486986
A1 David C. Wych
A1 Phillip C. Aoto
A1 Lily Vu
A1 Alexander M. Wolff
A1 David L. Mobley
A1 James S. Fraser
A1 Susan S. Taylor
A1 Michael E. Wall
YR 2022
UL http://biorxiv.org/content/early/2022/04/05/2022.04.04.486986.abstract
AB To assess the potential benefits of molecular-dynamics (MD) simulations for macromolecular crystallography (MX), we performed room-temperature X-ray diffraction studies of the catalytic subunit of mouse protein kinase A (PKA-C). We then performed crystalline MD simulations of PKA-C, computed simulated electron densities from the water, protein, and ion components of the MD simulations, and carefully compared them to the initial crystal structure. The results led to the development of an MD-MX analysis procedure and several associated methods: 1) density comparison to evaluate consistency between the MD and the initial crystal structure model; 2) water building to generate alternative solvent models; and 3) protein remodeling to improve the crystal structure where interpretation of density is unclear. This procedure produced a revised structure of PKA with a new ordered water model and a modified protein structure. The revisions yield new insights into PKA mechanisms, including: a sensitivity of the His294 conformation to protonation state, with potential consequences for regulation of substrate binding; a remodeling of the Lys217 side chain along with a bound phosphate; an alternative conformation for Lys213 associated with binding to the regulatory subunit; and an alternative conformation for catalytic base Asp166 and nearby waters, suggesting a mechanism of progression of the phosphotransfer reaction via changes in Mg2+ coordination. Based on the benefits seen applying these methods to PKA, we recommend incorporating our MD-MX procedure into MX studies, to decide among ambiguous interpretations of electron density that occur, inevitably, as part of standard model refinement.Competing Interest StatementPCA is currently an employee of Eli Lilly and Company. DLM is on the Scientific Advisory Board of OpenEye Scientific Software and is an Open Science Fellow with Roivant. JSF is a consultant for, has equity in, and receives research support from Relay Therapeutics.