Force Distribution Analysis of Allosteric Mechanisms

Abstract

Revealing the pathways of signal transfer in allosteric proteins has remained a challenge for today's biophysical methods. Previous approaches are primarily based on the comparison of active and inactive structures and thermodynamic concepts. However, stiff regions of the protein might mask signal propagation, even though they are able to carry signals in form of high internal stresses.We here present a new method, force distribution analysis(FDA Stacklies et al,2009a/b), that detects the distribution of stress upon external perturbations in macromolecules like proteins, other (bio-)polymers or even solids with high sensitivity. For tracing signal transfer through a protein structure, FDA calculates the changes, here caused by ligand binding, in the inter-atomic forces of the protein as sampled in Molecular Dynamics simulations.The analyzed proteins are two homologues of the chaperone Hsp90 and the catabolite activator protein CAP.We propose a new model for the signal transduction in the E.Coli(HtpG) and Yeast(Hsp82) homologues of Hsp90. The force differences between the apo, ADP and ATP bound states obtained by FDA based on all-atom trajectories totalling 540 ns revealed a cross-talk between the binding site and the middle domain via distinct paths.The catabolite activator protein(CAP) is a major player in the lac-operon. CAP features a negative cooperativity for the binding of cAMP, which is interestingly based not on a change in structure but in flexibility (Popovych et al.,2006). FDA of the apo, single- and double-bound state revealed a signaling network in CAP, which transfers a signal (first cAMP is bound) to the second binding niche without obvious structural changes, thereby explaining the observed cooperativity.This work describes the effectiveness of FDA for resolving allosteric communication pathways, which are directly testable by experiments. As such, it has broad implications for our view on protein internal strain and function.