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

Abstract

Ligand binding and conformational changes of biomacromolecules play a central role in the regulation of cellular processes. It is important to understand how both are coupled and what their role is in biological function. The biochemical properties, conformational states, and structural dynamics of periplasmic substrate-binding proteins (abbreviated SBPs or PBPs), which are associated with a wide range of membrane proteins, have been extensively studied over the past decades. Their ligand-binding mechanism, i.e., the temporal order of ligand-protein interactions and conformational changes, however, remains a subject of controversial discussion. We here present a biochemical and biophysical analysis of the E. coli glutamine-binding protein GlnBP concerning ligand binding and its coupling to conformational changes. For this, we used a combination of experimental techniques including isothermal titration calorimetry, single-molecule Förster resonance energy transfer, and surface-plasmon resonance spectroscopy. 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. Furthermore, we also demonstrate that ligand binding and conformational changes in GlnBP are highly correlated. A global analysis of our results is consistent with a dominant induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, we suggest that the rigorous experimental and theoretical framework used here can be applied to other protein systems where the coupling mechanism of conformational changes and ligand binding is yet unclear or where doubts prevail.