Effects of ligand binding on the rigidity and mobility of proteins : a computational and experimental approach

The interplay between protein structure, flexibility, mobility and function is a central concern in structural biology. Here, we have studied the interactions of two different proteins, HIV-1 protease and cyclophilin A (CypA), with a variety of ligands. HIV-1 protease is a key modern drug target due to its role in the lifecycle of the virus HIV-1. CypA is a multifunctional protein which acts principally as an enzyme during protein folding, but also as the primary binding partner for the immunosuppressant drug cyclosporin A (CsA). Using a computational approach we have studied the manner in which different drugs affect the flexibility of HIV-1 protease. We have used experimental and computational approaches to investigate the binding effect of CsA on the structure, flexibility and mobility of CypA. The wealth of structural data available, particularly from X ray crystallography studies, creates an opportunity for computational scientists to provide data on flexibility and mobility in order to augment the structural data and inform future experiments. We use rapid computational tools to model the flexibility (first) and mobility (froda) with a variety of structural data as input. We characterised, with first, the effect of ligand binding on the rigidity of more than 200 X-ray crystal structures of HIV-1 protease. A similar approach has been applied to the structural dataset available for CypA. In addition, we have used froda to simulate the mobility of the protein. A new procedure, of tracking surface exposure during large-scale simulations of protein motion and combining the information with rigidity analysis data, was used to try to predict results of hydrogen-deuterium exchange NMR experiments (HDX). Experimentally, we have characterised CypA and its binding with CsA and subsequently performed NMR spectroscopy including HDX on both the unliganded CypA and the CypA-CsA complex. Small changes in chemical shift and the HDX folding core were observed upon ligand binding. This is the first study of the CypA-CsA complex using HDX. Our method for predicting the results of HDX for CypA is an improvement on the first-only approach, but we find that it is not yet sufficiently sensitive to predict the effects of ligand binding on CypA using these experiments. It is hoped that this work will contribute to the general understanding of ligand interactions, the particular interactions involving HIV-1 protease and CypA, and the applications of computational simulation using rigidity analysis and large-scale coarse-grained motion.