Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump.

Xin Xu,Song Xiang,Ying Gao,Pu Chen,Xinzheng Zhang,Huigang Shi,Xiaowen Gong

doi:10.7554/elife.53853

Abstract

The oxaloacetate decarboxylase sodium pump (OAD) is a unique primary-active transporter that utilizes the free energy derived from oxaloacetate decarboxylation for sodium transport across the cell membrane. It is composed of 3 subunits: the α subunit catalyzes carboxyl-transfer from oxaloacetate to biotin, the membrane integrated β subunit catalyzes the subsequent carboxyl-biotin decarboxylation and the coupled sodium transport, the γ subunit interacts with the α and β subunits and stabilizes the OAD complex. We present here structure of the Salmonella typhimurium OAD βγ sub-complex. The structure revealed that the β and γ subunits form a β3γ3 hetero-hexamer with extensive interactions between the subunits and shed light on the OAD holo-enzyme assembly. Structure-guided functional studies provided insights into the sodium binding sites in the β subunit and the coupling between carboxyl-biotin decarboxylation and sodium transport by the OAD β subunit.

Highlights

The sodium gradient across the cell membrane is utilized in many microorganisms as an energy source to drive ATP synthesis, nutrient intake, cell motility and other processes (Mulkidjanian et al, 2008)
We initially attempted to study the structure of the Salmonella typhimurium OAD (StOAD) holoenzyme and expressed it in Escherichia coli (E. coli) and purified it through a 6x Histidine tag engineered to the N-terminus of the g subunit and nickel-nitrilotriacetic acid (Ni-NTA)
Dynamic light scattering experiments indicated that it has a molecular weight of 250 kDa, suggesting that it is suitable for cryo-electron microscopy (EM) studies

Summary

Introduction

The sodium gradient across the cell membrane is utilized in many microorganisms as an energy source to drive ATP synthesis, nutrient intake, cell motility and other processes (Mulkidjanian et al, 2008). The oxaloacetate decarboxylase sodium pump (OAD) is the first identified sodium pump responsible for maintaining the sodium gradient in microorganisms (Dimroth, 1980) It is found in many bacteria and archaea including the human pathogens Klebsiella aerogenes (Dimroth, 1980), Klebsiella pneumonia (Schwarz et al, 1988), Salmonella typhimurium (Wifling and Dimroth, 1989) and Legionella pneumophila (Jain et al, 1996). OAD utilizes the free energy derived from oxaloacetate decarboxylation to drive sodium transport across the cell membrane. It is composed of a, b and g subunits. The carboxyltransferase (CT) domain in the a subunit catalyzes the carboxyl-transfer from oxaloacetate to biotin; the b subunit catalyzes the subsequent carboxyl-biotin decarboxylation and the coupled sodium transport; the g subunit stabilizes the OAD complex by interacting with the a and b subunits (Buckel, 2001; Dimroth et al, 2001)

Methods

Results

Conclusion