A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin
Figure 5
Interaction of Pga7 and Rbt5 with heme.
(A) Recombinant wild type Rbt523–219 and Pga718–195 or their mutants in a conserved aspartic acid residue were incubated with hemin agarose (Hm) or glutathione agarose (glut) beads. The beads were washed and bound proteins were released by heating to 100°C in SDS gel loading buffer, separated by PAGE and detected by Western blotting with an anti-Myc antibody. (B) ITC analysis of the interaction of hemin with Rbt523–219 (left panel), and with Pga718–195 (right panel). The proteins (60 µM) were titrated into 10 µM hemin (Pga7) or into 20 µM hemin (Rbt5). Representative experiments are shown, with the heat signal in the top half of each panel and the binding isotherm derived from this signal in the lower half. The average fitting of three independent experiments with Rbt5-heme gave n = 2.152±0.0706 sites, Ka = 1.852×105±3.679×104 M−1 and H = −7454±304.7 KJ mol−1. The average fitting of three independent experiments with Pga7-heme gave n = 1.930±0.00999 sites, Ka = 1.531×107±1.817×106 M−1 and H = −7002±56.69 KJ mol−1. (C) UV/visible spectra from the titration of 1 µl hemin aliquots (2 mM hemin stock) into 3 ml of 5.5 µM apoPga718–195 (1 cm path length cuvette, PBS). The inset shows the hemin-Pga7 absorbance minus hemin alone absorbance at 406 nm, at increasing hemin concentrations. The inflection point indicates saturation of hemin binding to Pga7 at close to 1∶1 concentration.