Escherichia coli CorA Periplasmic Domain Functions as a Homotetramer to Bind Substrate

Shi-Zhen Wang,Yong Chen,Zhan-Hua Sun,Qiang Zhou,Sen-Fang Sui

doi:10.1074/jbc.m602342200

Abstract

CorA is a primary Mg2+ transporter in bacteria, which also mediates influx of Ni2+ and Co2+. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning alpha-helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-PPD indicated that E. coli CorA PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full-length CorA and PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.

Highlights

Three magnesium transport systems have been identified in bacteria: CorA, MgtA/B, and MgtE (9 –15)
Residues, with a unique topology [21]. It can be considered as a two-domain protein: a 235-amino acid residue N-terminal-soluble domain, which is located in the periplasmic space (periplasmic domain (PPD)3), followed by a transmembrane domain (TM) composed of three transmembrane segments [21]
Typhimurium CorA, which has only an 8-amino acid residue difference compared with E. coli CorA, the CorA PPD was expressed by the encoding region from Met-1 to Ile223 in E. coli

Summary

Introduction

Three magnesium transport systems have been identified in bacteria: CorA, MgtA/B, and MgtE (9 –15). CorA is the first identified gene mediating Mg2ϩ influx [16, 17] and by far the most abundant prokaryotic magnesium transporter (18 – 20). It shares no sequence homology with any other known membrane protein or transporter families [10, 21]. Primary structural and functional studies indicate that CorA is unusual among secondary transporters It expresses constitutively and transports Mg2ϩ with high capacity. CorA is the most widely studied magnesium transporter in bacteria, little is known about its structural and molecular roles of the large N-terminal PPD in Mg2ϩ transport. The pyramid-like homotetramer of CorA PPD with a central cavity may connect with the “magnesium pore,” forming a “tunnel” that mediates ion transport

Methods

Results

Conclusion