Figure 1.
P. aeruginosa PilY1 strains contain conserved integrin binding residues RGD and conserved putative calcium binding site.
(a) A bar representation of PilY1. The consensus c-terminal pilus biogenesis domain is in blue and the green represents the n-terminal addition to the previously examined construct. Calcium binding motifs are highlighted in yellow and the RGD is highlighted in orange. (b) Five varying strains of P. aeruginosa PilY1 were aligned using the biology workbench server [26], [27]. Blue residues and an “*” corresponds to identical residues throughout the 5 strains, green residues and a “:” correspond to highly conserved residues, and navy residues and a “.” corresponds to mildly conserved residues (e.g. alanine and leucine). Reference numbers are for PAK_287 PilY1 (the strain used in this study).
Figure 2.
Purified PilY1 binds to integrin in an RGD dependent manner.
(a) PilY1 was titrated onto a solid phase binding assay keeping the concentration of αVβ5 integrin constant at 2 µg/mL. The curve was fit to the ligand binding, one site saturation equation f = Bmax*abs(x)/(Kd+abs(x)) with an r2 = 0.98. The Kd was calculated at 164 nM±38. (b–d) Wild type PilY1 was established as a reference point and the value all measurements were compared to. (b) RGDS inhibitor peptide or GRADSP control peptide were added in 2, 100, and 250 fold molar excess to PilY1. “*” corresponds to p<.1 compared to PilY1 binding to αVβ5 integrin. (c) PilY1 was added with or without 50 fold molar excess of the inhibitor RGDS peptide to 2 µg/mL of αVβ3 integrin. “**” corresponds to p<.02 compared to PilY1 binding to αVβ3 integrin. (d) ΔRGD (Δ619–621) or D621A mutations of PilY1 were added to 2 µg/mL of αVβ5 integrin. “***” corresponds to p<.01 compared to PilY1 binding to αVβ5 integrin. (e) Molar ellipticity values were calculated for the respective wavelength scans and compared.
Figure 3.
PilY1 has two functional calcium binding sites.
(a) An alignment of the two calcium binding sites in PilY1 was performed as in figure 1. Calcium coordinating residues are underlined. (b, c, and d) A calcium competition binding assay using Oregon Green was performed with (b) D859A and D859K, (c) D608A and D608K, and (d) wild type and D608A/D859A. Binding curves were modeled to one-site competition (D859A, D859K, D608A, D608K, and wild type), or linear line (D608A/D859A). Error represents standard error of the mean.
Figure 4.
Calcium binding sites play a role in PilY1's ability binds to integrin.
(a) D608A, D608K, D859A, D859K (b) D608A/D859A, D608A/D859K, D608K/D859A, and D608K/D859K mutations of PilY1 were added to αVβ5 integrin as seen in figure 2B. “*” corresponds to p<.01 compared to wild type PilY1 binding to αVβ5 integrin and “**” corresponds to p<.1 as compared to wild type PilY1 binding to αVβ5 integrin. (c) This model represents the calcium binding effects on PilY1 binding to integrin. D to A mutations represent mutations in the final aspartic acid of the calcium binding site.
Figure 5.
Calcium effects on RGD binding proteins.