A cationic lumen in the Wzx flippase mediates anionic O-antigen subunit translocation in Pseudomonas aeruginosa PAO1.

Molecular Microbiology
Salim T IslamJoseph S Lam

Abstract

Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS). Herein, site-directed mutagenesis targeting 102 residues was carried out on the TMS and loops of Wzx, followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function. The importance of various charged and aromatic residues was highlighted, predominantly within the TMS of the protein, revealing functional 'hotspots' within the flippase, particularly within TMS2 and TMS8. Construction of a tertiary structure homology model for Wzx indicated that TMS2 and TMS8 line a central cationic lumen. This is the first report to describe a charged flippase lumen for mediating anionic O-unit translocation across the hydrophob...Continue Reading

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Citations

Nov 2, 2014·Canadian Journal of Microbiology·Salim T Islam, Joseph S Lam
Apr 23, 2014·Nucleic Acids Research·Marcus StammLucy R Forrest
Nov 2, 2015·Seminars in Cell & Developmental Biology·Salim T Islam, Tâm Mignot
Sep 29, 2012·Environmental Microbiology·Salim T Islam, Joseph S Lam
Mar 13, 2014·FEMS Microbiology Reviews·Sergei KalynychMiroslaw Cygler
Jan 23, 2016·Lipid Insights·Natividad Ruiz
Apr 16, 2015·Chembiochem : a European Journal of Chemical Biology·Jochen Schmid, Volker Sieber
Sep 17, 2014·Journal of Bacteriology·Emily K ButlerNatividad Ruiz
Sep 16, 2015·Journal of Bacteriology·Gaëlle S KollyStewart T Cole
Jun 23, 2020·Annual Review of Biochemistry·Christopher A CaffaletteJochen Zimmer
Apr 17, 2019·Nature Communications·Alvin C Y KukSeok-Yong Lee
Jun 10, 2020·PLoS Biology·Salim T IslamEmilia M F Mauriello
Aug 30, 2021·Molecular Microbiology·Fares SaïdiSalim T Islam

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Datasets Mentioned

BETA
AE003852

Methods Mentioned

BETA
X-ray

Software Mentioned

Quantity One
MEMPACK
HHpred
MUSCLE
PyMol
NorM
TM
MODELLER
SHARCNET
hit

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