Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding

Abstract

Staphylococci use cell wall-anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall-anchored protein, uro-adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain-truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus.

Figure. 1

Domain organization of UafA and partial proteins used for structure determination. S, signal peptide; A, A region; N1, N2, N3, domains in the A region; B, B region of about 20 kDa; R, region of low sequence complexity. Numbers indicate the residue number of each position.

Figure. 2

Hemagglutination inhibition activity of UafA-F^{50 kDa}. Erythrocytes were incubated with S. saprophyticus cells and 2-fold serially diluted UafA-F^{50 kDa}. As controls, erythrocytes without S. saprophyticus cells and UafA-F^{50 kDa} are also shown.

Figure. 3

Crystal structure of functional region of UafA. (A) Overall structure of UafA-F^(392-811). N2, N3, and B domains are shown in green, orange, and red, respectively. The N3-B loop is shown as a gray loop. (B) Topology diagrams of UafA^(392-811). Arrows and boxes represent β-strands and α-helixes, respectively. The colors correspond to those in Figure 3(A). (C) Superposition of three structures. UafA-F^(376-811), P2₁ form UafA-F^(392-811) (1.5 Å resolution), and P2₁2₁2₁ form UafA-F^(392-811) (1.7 Å resolution) are shown in blue, red, and green, respectively. Cα atoms in N3 domains are superposed.

Figure. 5

Structure comparison of the B domain of UafA with that of CNA. (A) Stereo diagram of the B domain of UafA superposed onto that of CNA. Gly694–Asp809 of UafA (red) and Ser538–Thr623 of CNA (green) are superposed. Potassium ions bound in the B domain of UafA are also shown as red balls. (B) Residues absent in CNA or of which Cα are more than 4 Å apart from the position of the corresponding residue in CNA are shown in red. The bound potassium atoms are also shown as green balls.

Figure. 4

Stereo diagram of the superposed B domains in UafA-F^(376-811), P2₁ form UafA-F^(392-811), and P2₁2₁2₁ form UafA-F^(392-811). The colors correspond to those in Figure 3(C). The bound potassium ions are also shown as red balls.

Figure. 6

Hemagglutination inhibition activity of the mutants. (A) Domain organization of each mutant prepared for functional analysis. The N3-B loop is shown as a gray box. (B) Hemagglutination inhibition activity of each mutant. Erythrocytes were incubated with S. saprophyticus cells and 100 μg of the truncated protein. Positive control, erythrocytes with S. saprophyticus cells; Negative control, erythrocytes without S. saprophyticus cells or UafA.

Figure. 7

Hypothetical ligand binding model of UafA. The ligand binding mechanism of UafA (A, present study) and that of CNA (B16) are shown. N2, N3, and B domains are shown in green, yellow, and red, respectively. Collagen, which is a ligand of CNA, is also shown as a purple bar. The domain organization of each protein is also shown.