The Hemophilus influenzae Hap autotransporter is a chymotrypsin clan serine protease and undergoes autoproteolysis via an intermolecular mechanism.
The Hemophilus influenzae Hap adhesin is an autotransporter protein that undergoes an autoproteolytic cleavage event resulting in extracellular release of the adhesin domain (Hap(s)) from the membrane-associated translocator domain (Hap(beta)). Hap autoproteolysis is mediated by Ser(243) and occurs at LN1036-7 and to a lesser extent at more COOH-terminal alternate sites. In the present study, we sought to further define the mechanism of Hap autoproteolysis. Site-directed mutagenesis of residues His(98) and Asp(140) identified a catalytic triad conserved among a subfamily of autotransporters and reminiscent of the SA (chymotrypsin) clan of serine proteases. Amino-terminal amino acid sequencing of histidine-tagged Hap(beta) species and site-directed mutagenesis established that autoproteolysis occurs at LT1046-7, FA1077-8, and FS1067-8, revealing a consensus target sequence for cleavage that consists of ((Q/R)(A/S)X(L/F)) at the P4 through P1 positions. Examination of a recombinant strain co-expressing a Hap derivative lacking all cleavage sites (HapDelta1036-99) and a Hap derivative lacking proteolytic activity (HapS243A) demonstrated that autoproteolysis occurs by an intermolecular mechanism. Kinetic analysis of Hap autoproteolysis in bacteria expressing Hap under control of an inducible promoter demonstrated that autoproteolysis increases as the density of Hap precursor in the outer membrane increases, confirming intermolecular cleavage and suggesting a novel mechanism for regulation of bacterial adherence and microcolony formation.
Fink
D L
DL
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Cope
L D
LD
Hansen
E J
EJ
Geme
J W
JW
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology*; D001665:Binding Sites; D001692:Biological Transport; D002384:Catalysis; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000737:chemistry*; D017124:Conserved Sequence; D005821:Genetic Techniques; D006193:Haemophilus influenzae / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; C091552:Hap protein, Hemophilus influenzae; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D012697:Serine Endopeptidases; D002918:Chymotrypsin