title: Structural determinants of autoproteolysis of the Haemophilus influenzae Hap autotransporter.
abstract: Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapbeta C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.
authors.affiliation: Department of Pediatrics, Duke University, Durham, NC 27710, USA.
authors.forename: Roma
authors.initials: R
authors.lastname: Kenjale
authors.forename: Guoyu
authors.initials: G
authors.lastname: Meng
authors.forename: Doran L
authors.initials: DL
authors.lastname: Fink
authors.forename: Twyla
authors.initials: T
authors.lastname: Juehne
authors.forename: Tomoo
authors.initials: T
authors.lastname: Ohashi
authors.forename: Harold P
authors.initials: HP
authors.lastname: Erickson
authors.forename: Gabriel
authors.initials: G
authors.lastname: Waksman
authors.forename: Joseph W
authors.initials: JW
authors.lastname: St Geme
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C091552:Hap protein, Hemophilus influenzae; D012697:Serine Endopeptidases
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