Biophysical Characterization of Interaction Between Domain A and B of the Mannitol Transporter Enzyme II by NMR Spectroscopy

Abstract

In multi-domain proteins, protein linkers serve to maintain separate domains within a certain distance, and also affect the domain-domain motion. The cytoplasmic A and B domains of mannitol transporter enzymes are well-conserved across bacterial strains. Domain A and B are covalently connected by a flexible linker in Escherichia coli, whereas they are separated in the thermophilic bacterium Thermoanaerobacter Tengcongensis (Tt). In this study, I investigated changes in domain association changes when the domains have evolved with or without their connecting linkers by characterizing the structure and interaction of the Tt mannitol transporter A (TtIIAmtl) and phosphorylated B (phospho-TtIIBmtl) domains. The backbone folds and interface of individual domains were well conserved between the two bacterial strains. However, separated domains showed 28 times higher affinity than the linked ones. The increase in binding affinity mainly resulted from different compositions of interfacial residues, with the general backbone fold of the complex remaining unperturbed. Phosphorylation of the active site loop significantly contributed to the binding affinity by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were sufficient to account for the enhanced binding affinity. Specifically, the complex was stabilized by extended hydrophobic interactions through T542I and P543T and from hydrogen bonds between the hydroxyl group of V557S and the carboxyl backbone group of A386, which enhances the affinity thereby enabling a proper domain association regardless of the presence of the linker. Interestingly, the population of the association state in vivo was estimated to be nearly identical between the non-linked and linked domains. These results suggest that the binding affinity between domains is, modulated in response to changes in the connecting linkers, which would help to maintain the domain association at a functional level regardless of the presence of the linker.