Verification of a topology model of PorT as an integral outer-membrane protein in Porphyromonas gingivalis.
PorT is a membrane-associated protein shown to be essential for the maturation and secretion of a class of cysteine proteinases, the gingipains, from the periodontal pathogen Porphyromonas gingivalis. It was previously reported that PorT is located on the periplasmic surface of the inner membrane to function as a chaperone for the maturing proteinases. Our modelling suggested it to be an integral outer-membrane protein with eight anti-parallel, membrane-traversing beta-strands. In this report, the outer-membrane localization model was confirmed by the structural and functional tolerance of PorT to hexahistidine (6xHis) tag insertions at selected locations within the protein using site-directed mutagenesis. Interestingly, those PorT mutations adversely affecting gingipain secretion enhanced expression of the porT gene but at the same time suppressed the transcription of the gingipain rgpB gene. Further, PorT mutants deficient in gingipain activities produced significantly more di- and triaminopeptidase activities. PorT homologues have been found in restricted members of the Bacteroidetes phylum where there is potential for PorT to participate in the maturation and secretion of proteins with characteristic C-terminal domains (CTDs). Knowledge of the cellular localization of PorT will enable analysis of the role of this protein in a new secretory pathway for the export of gingipains and other CTD-class proteins.
Nguyen
Ky-Anh
KA
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.|Institute of Dental Research, Westmead Centre for Oral Health and Westmead Millennium Institute, Sydney, Australia.
Żylicz
Jasiek
J
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Szczesny
Pawel
P
Department of Bioinformatics, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
Sroka
Aneta
A
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Hunter
Neil
N
Institute of Dental Research, Westmead Centre for Oral Health and Westmead Millennium Institute, Sydney, Australia.
Potempa
Jan
J
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.|Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D001425:Bacterial Outer Membrane Proteins; D003546:Cysteine Endopeptidases