Carbohydrate Affinity for the Glucose–Galactose Binding Protein Is Regulated by Allosteric Domain Motions

Abstract

Protein function, structure, and dynamics are intricately correlated, but studies on structure–activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: <i>Escherichia coli</i> glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an <i>induced fit</i> mechanism; on the other hand, GGBP shows extensive segmental mobility in both <i>apo</i> and <i>holo</i> states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a <i>population shift</i> mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding