title: Topological analysis of the Escherichia coli WcaJ protein reveals a new conserved configuration for the polyisoprenyl-phosphate hexose-1-phosphate transferase family.
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 data demonstrate a novel topological configuration for PHPTs, which is proposed as a signature for all members of this enzyme family.
authors.affiliation: Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Furlong
authors.affiliation: Centre for Infection and Immunity, Queen's University Belfast, Belfast, United Kingdom, BT9 7AE.
authors.forename: Amy
authors.initials: A
authors.lastname: Ford
authors.affiliation: Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
authors.forename: Lorena
authors.initials: L
authors.lastname: Albarnez-Rodriguez
authors.affiliation: 1] Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada [2] Centre for Infection and Immunity, Queen's University Belfast, Belfast, United Kingdom, BT9 7AE.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C573281:WcaJ protein, E coli