R6G narrows BmrA conformational spectrum for a more efficient use of ATP

Abstract

Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to change conformation and thereby translocate substrates out of the cell to detoxify them. While this general access mechanism scheme is well accepted, molecular details of this interplay is still elusive. Rhodamine6G binding on a catalytic mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise Michaelian. We investigated this asymmetric behavior via a structural-enzymology approach, solving cryoEM structure of BmrA at defined ATP ratio along the enzymatic transition, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, revealed that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding-domains, describing the allosteric effect of drug binding that optimizes the ATP-dependent conversion of the transporter to the outward-facing state. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the allosteric transition occurs. Drug diffusion rate is the likely rate-limiting step of the reaction, while drug transport and ATPase activities are in effect uncoupled.