Crystal Structures of the Choline/Acetylcholine Substrate-binding Protein ChoX from Sinorhizobium meliloti in the Liganded and Unliganded-Closed States

Christine Oswald,Sander H.J Smits,Marina Höing,Linda Sohn-Bösser,Laurence Dupont,Daniel Le Rudulier,Lutz Schmitt,Erhard Bremer

doi:10.1074/jbc.m806021200

Abstract

The ATP-binding cassette transporter ChoVWX is one of several choline import systems operating in Sinorhizobium meliloti. Here fluorescence-based ligand binding assays were used to quantitate substrate binding by the periplasmic ligand-binding protein ChoX. These data confirmed that ChoX recognizes choline and acetylcholine with high and medium affinity, respectively. We also report the crystal structures of ChoX in complex with either choline or acetylcholine. These structural investigations revealed an architecture of the ChoX binding pocket and mode of substrate binding similar to that reported previously for several compatible solute-binding proteins. Additionally the ChoX-acetylcholine complex permitted a detailed structural comparison with the carbamylcholine-binding site of the acetylcholine-binding protein from the mollusc Lymnaea stagnalis. In addition to the two liganded structures of ChoX, we were also able to solve the crystal structure of ChoX in a closed, substrate-free conformation that revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. This structure is only the second of its kind and raises the important question of how ATP-binding cassette transporters are capable of distinguishing liganded and unliganded-closed states of the binding protein.

Highlights

Fixation of nitrogen and its delivery to its host plant Medicago sativa [2], uses choline as the sole carbon and nitrogen source [1]
In the case of the ABC importer ChoVWX from S. meliloti, ChoV serves as the nucleotide-binding domain that provides energy for the transport process via ATP hydrolysis, ChoW is the integral cytoplasmic membrane protein that forms the substrate translocation pathway, and ChoX is the substrate-binding protein located in the periplasm [8]
The data reported by Dupont et al [8] already suggested that the ChoVWX ABC transporter is capable of catalyzing the uptake of both choline and ACh

Summary

Introduction

Fixation of nitrogen and its delivery to its host plant Medicago sativa [2], uses choline as the sole carbon and nitrogen source [1]. We were able to solve the crystal structure of ChoX in a substrate-free closed conformation with an architecture of the ligand-binding site that is superimposable for the closed ligand-bound and closed-unliganded forms of ChoX.

Results

Conclusion