Integrating Protein Dynamics And Enhanced Conformational Ensembles To Better Understand Their Role In Biomolecular Function

Proteins have been termed the building blocks of life due to their involvement in practically every biological process that occurs in a living organism. For years now, researchers have sought to uncover the underlying mechanisms employed by biomolecules to carry out such tasks and functions. Initial understanding of proteins and how they function, at the atomic level, was revolutionized by the generation of numerous average static structures via X-ray crystallographic methods. Nevertheless, although one or multiple three-dimensional structures exist for many proteins, biological activity cannot be solely explained by relatively rigid structures. Proteins are dynamic entities governed by their dynamic personalities where biological function is rooted in their internal motions, fluctuations, and conformational changes. However, despite many experimental and computational efforts, how protein motions or dynamics couple protein function remains poorly understood. Here, in this work, we employ standard molecular dynamics (MD) simulations and enhanced sampling methods (Rotatable accelerated MD-dual boost (RaMD-db)) to try and capture a more accurate representation of the dynamic nature of two enzymes, cyclophilin A and choline oxidase. We show that molecular dynamics is a powerful method and is more than capable of acquiring a more accurate representation of the dynamic nature of the enzymes, in comparison to experimental techniques. More so, RaMD-db, because it was able to sample conformational states that were never observed in standard MD. Furthermore, we showed, at an atomic level, how protein motions facilitate and are coupled to biological function in both cyclophilin A (CypA) and choline oxidase. Ultimately, an atomic level description of how protein motions facilitate function, provided by the results in this work, can be utilized for drug design advancement, protein engineering, and to gain a better understanding of protein participation in disease

Using Molecular Dynamics to Elucidate the Mechanism of Cyclophilin

Cyclophilins are ubiquitous enzymes that are involved in protein folding, signal transduction, viral proliferation, oncogenesis, and regulation of the immune system. Cyclophilin A is the prototype of the cyclophilin family. We use molecular dynamics to describe the catalytic mechanism of cyclophilin A in full atomistic detail by sampling critical points along the reaction coordinate, and use accelerated molecular dynamics to sample cis-trans interconversions. At these critical points, we analyze the conformational space sampled by the active site, flexibility of the enzyme backbone, and modulation of binding interactions.We use Kramer’s rate theory to determine how diffusion and free energy contribute to lowering the activation energy of prolyl isomerization. We also find preferential binding modes of several cyclophiln A inhibitors, and compare the conformational space sampled by inhibited cyclophilin A to the conformational space sampled during wild-type interactions. We also analyze the mechanism of the next family member cyclophilin B in order to probe differences in enzyme dynamics and intermolecular interactions that could possibly be exploited in isoform-specific drug design. Our results indicate that cyclophilin proceeds by a conformational selection binding mechanism that manipulates substrate sterics, electrostatic interactions, and multiple reaction timescales in order to speed up reaction rate. Conformational space sampled by cyclophilin when inhibited and when undergoing wild-type interactions share significant similarity. Cyclophilins A and B do have notable differences in enzyme dynamics, due to variation in intramolecular interactions that arise from variation in primary structures. This work demonstrates how computational methods can be used to clarify catalytic mechanisms