title: Pseudomonas aeruginosa PilY1 binds integrin in an RGD- and calcium-dependent manner.
abstract: PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850-859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619-621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600-608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851-859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Michael D L
authors.initials: MD
authors.lastname: Johnson
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Garrett
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Bond
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Coggan
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Wolfgang
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Redinbo
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D017124:Conserved Sequence; D036781:Fimbriae Proteins / Q000737:chemistry / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D016023:Integrins; D009842:Oligopeptides; C103855:PilY1 protein, Pseudomonas aeruginosa; D036781:Fimbriae Proteins; C047981:arginyl-glycyl-aspartic acid; D002118:Calcium