Ligand-induced conformational changes in a thermophilic ribose-binding protein.

Matthew J Cuneo,Homme W Hellinga,Lorena S Beese

doi:10.1186/1472-6807-8-50

Matthew J Cuneo, Homme W Hellinga + Show 1 more

Open Access

https://doi.org/10.1186/1472-6807-8-50

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Abstract

BackgroundMembers of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.ResultsHere we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (appTm value is 108°C) than the mesophilic Escherichia coli homolog (ecRBP) (appTm value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.ConclusionAlthough the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.

Highlights

Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes
Expression The ribose-binding protein (RBP) gene was identified in the T. maritima genome sequence [17] as open reading frame (ORF) tm0958, based on sequence similarity to the E. coli RBP, and genetic linkage of this ORF within a putative operon that contains sequences for ABC transporters characteristic of a ribose transport system [18]
The nucleotide sequence of the recombinant was confirmed by DNA sequencing. Overexpression of this ORF in E. coli produced ~50 mg of pure protein per liter of growth medium, which was purified by immobilized metal affinity chromatography [19] followed by gel filtration chromatography

Summary

Introduction

Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Solute binding induces a conformational change to form a closed state in which the ligand is bound at the domain interface and buried by the surrounding protein [6-8]. This closed form typically binds to other molecular components to trigger downstream cellular processes such as chemotaxis [9], quorum sensing [3], and transmembrane ligand transport [10]. Eukaryotic receptors that contain the PBP fold as part of multi-domain proteins are regulated by ligand-induced conformational coupling mechanisms [11]

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