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Poster Abstract – Erice 2011
Transmembrane domain charge character is required for function of the O-antigen
flippase Wzx in Pseudomonas aeruginosa PAO1
Salim T. Islam1, Erin A. Anderson1, Robert C. Ford2, and Joseph S. Lam1
1
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G
2W1, Canada
2
Faculty of Life Science, University of Manchester, Manchester, M50 1QD, United Kingdom
The Wzy-dependent pathway for the biosynthesis of cell-surface polysaccharides is
applicable to a wide range of Gram-negative bacteria, yet it remains poorly understood. In
Pseudomonas aeruginosa, an often fatal opportunistic pathogen of compromised patients, this
pathway is responsible for the synthesis of the immunodominant lipopolysaccharide glycoform,
capped by a negatively-charged heteropolymeric O antigen (O-Ag). Initial translocation of
trisaccharide O-Ag subunits bound to undecaprenyl pyrophosphate, from the inner leaflet to the
outer leaflet of the inner membrane (IM), is believed to be mediated by the O-Ag flippase Wzx,
an integral IM protein. While Wzx proteins are found in a wide range of bacteria, structural data
to explain their purported function was non-existent until a recent investigation by our group in
which the detailed topology of Wzx from P. aeruginosa PAO1 was mapped. This study revealed
the presence of 12 transmembrane segment (TMS) helices containing a range of charged amino
acids within the membrane-embedded portion of the protein1.
To gain a better understanding of the structure and function of Wzx, we have initiated
various genetic, biochemical, and biophysical studies. TMS helix-packing arrangements using
molecular dynamics data support the presence of a charged channel running down the length of
the protein, providing a plausible explanation for the mechanism of Wzx function. Moreover,
thorough site-directed mutagenesis analyses (> 100 aa) have confirmed the importance of
numerous charged amino acids within the TMS helices, with the corresponding mutants still able
to target to the IM but unable to complement a chromosomal wzx deficiency, further supporting
our hypothesis. Optimal overexpression and detergent solubilization conditions have been
developed by expressing Wzx with a cleavable C-terminal His-tagged green fluorescent protein
(GFP-His8) fusion, allowing for measurement of in vivo and in vitro GFP fluorescence2. Single
particle reconstruction from transmission electron microscopy (TEM) of negatively-stained
monodisperse populations of the protein has been carried out using the EMAN software suite to
gain preliminary tertiary structure insights. Wzx-GFP-His8 fusions have also been reconstituted
in membrane vesicles and verified by TEM as well as in-gel fluorescence scanning of SDSPAGE gels loaded with reconstituted vesicles. Trypsin-protection assays of vesicle-reconstituted
Wzx-GFP-His8 indicated that digestion of the vesicles did not yield mass ions that would
correspond to either Wzx or GFP when the enzymatic digests were analyzed by MALDI-TOF
MS, suggesting a preferred orientation of the construct within the vesicle, with the C-terminus of
Wzx (and the associated GFP tag) localized within the interior and the periplasmic face exposed
on the outside. This is consistent with the topology map we have generated as no periplasmic
loops contain trypsin-cleavage sites. Taken together, these analyses will greatly advance our
understanding of the structure and function of the flippase Wzx.
1
Islam et al., 2010. mBio 1(3):e00189-10
2
Drew et al., 2006. Nat. Methods 3(4):303-313