Topological analysis of the Escherichia coli WcaJ protein reveals a new conserved configuration for the polyisoprenyl-phosphate hexose-1-phosphate transferase family

Scientific Reports
Sarah E FurlongMiguel A Valvano

Abstract

WcaJ is an Escherichia coli membrane enzyme catalysing the biosynthesis of undecaprenyl-diphosphate-glucose, the first step in the assembly of colanic acid exopolysaccharide. WcaJ belongs to a large family of polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) sharing a similar predicted topology consisting of an N-terminal domain containing four transmembrane helices (TMHs), a large central periplasmic loop, and a C-terminal domain containing the fifth TMH (TMH-V) and a cytosolic tail. However, the topology of PHPTs has not been experimentally validated. Here, we investigated the topology of WcaJ using a combination of LacZ/PhoA reporter fusions and sulfhydryl labelling by PEGylation of novel cysteine residues introduced into a cysteine-less WcaJ. The results showed that the large central loop and the C-terminal tail both reside in the cytoplasm and are separated by TMH-V, which does not fully span the membrane, likely forming a "hairpin" structure. Modelling of TMH-V revealed that a highly conserved proline might contribute to a helix-break-helix structure in all PHPT members. Bioinformatic analyses show that all of these features are conserved in PHPT homologues from Gram-negative and Gram-positive bacteria. Our ...Continue Reading

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Citations

Aug 12, 2020·Journal of Bacteriology·María Pérez-BurgosLotte Søgaard-Andersen
May 18, 2018·Nature Chemical Biology·Leah C RayKaren N Allen
Dec 19, 2020·FEMS Microbiology Reviews·Tong SuLok-To Sham
Jan 22, 2021·Proceedings of the National Academy of Sciences of the United States of America·Katherine H O'TooleKaren N Allen
Dec 14, 2019·Cell Chemical Biology·Jean-Marie SwiecickiBarbara Imperiali
Jun 9, 2021·Biochemical Society Transactions·Katherine H O'TooleBarbara Imperiali

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Methods Mentioned

BETA
PCR
protein assay

Software Mentioned

Clustal Omega
TMHMM
DAS
Jalview
PSIPRED
ConSeq
TMKink
TMH

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