Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

Abstract

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.