Crystallization and preliminary X-ray diffraction analysis of YejM from Salmonella typhimurium: an essential inner membrane protein involved in outer membrane directed cardiolipin transport.

Uma Gabale,Gene Qian,Susanne Ressl,Elaina Roach

doi:10.12688/f1000research.8647.2

Uma Gabale, Gene Qian + Show 2 more

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https://doi.org/10.12688/f1000research.8647.2

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Journal: F1000Research	Publication Date: Dec 11, 2017
Citations: 1	License type: CC BY 4.0

Affiliation: Indiana University Bloomington

Abstract

Salmonella typhimuriumis responsible for over 35% of all foodborne illness related hospitalizations in the United States. This Gram-negative bacteriumpossesses an inner and an outer membrane (OM), the latter allowingits survival and replication within host tissues. During infection, OM is remodeled by transport of glycerophospholipids across the periplasm and into the OM. Increased levels of cardiolipin in the OM were observed upon PhoPQ activation and led to the discovery of YejM; an inner membrane protein essential for cell growth involved in cardiolipin binding and transport to the OM. Here we report how YejM was engineered to facilitate crystal growth and X-ray diffraction analysis. Successful structure determination of YejM will help us understand how they interact and how YejM facilitates cardiolipin transport to the OM. Ultimately, yejm, being an essential gene, may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium.

Highlights

YejM is comprised of 586 amino acids forming five predicted N-terminal transmembrane helices, followed by an arginine-rich periplasmic random coil linker region, and a C-terminal periplasmic domain (Figure 1A), and it was shown that YejM associates as a tetramer in solution (Dalebroux et al, 2015)
Initial crystals of YejM241-586 appeared after one week incubation at 18oC under different conditions; e.g. needle clusters in 2.8 M sodium acetate trihydrate pH 7.0, 0.1 M BIS-TRIS propane pH 7.0 (Hampton SaltRx condition A2, Hampton Research), needle clusters in 2.8 M sodium acetate (Hampton Index condition B12, Hampton Research), and rhombohedral crystals appeared in 3.5 M sodium formate pH 7.0 (Hampton Index HR conditions C1, Hampton Research)
We aim to solve the crystal structures of YejM alone and with bound cardiolipin. These structures will help in understanding where and how cardiolipin binds to YejM, and whether YejM’s architecture is that of a transporter or channel

Summary

Introduction

Salmonella typhimurium is a Gram-negative bacterium responsible for over 35% of all foodborne illness-related hospitalizations in the United States (Painter et al, 2013). The inner membrane protein YejM ( known as PbgA) was shown to bind cardiolipin and be involved in OM formation (Dalebroux et al, 2015). YejM is comprised of 586 amino acids forming five predicted N-terminal transmembrane helices, followed by an arginine-rich periplasmic random coil linker region, and a C-terminal periplasmic domain (Figure 1A), and it was shown that YejM associates as a tetramer in solution (Dalebroux et al, 2015). The arginine-rich linker region and periplasmic globular domain of YejM were demonstrated to bind cardiolipin and are required for OM remodeling and cell growth (Dalebroux et al, 2015). The molecular mechanism of the interplay between PhoPQ system and YejM, and how cardiolipin molecules are transported to the OM, need further structural and functional investigation

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