Abstract
BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 x 10(-5) m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.
Highlights
Tor-mediated endocytosis, and this event has likewise been demonstrated to suppress tumor necrosis factor-␣ responses and control of the infection [3]
BAD-1 consists of a 150-amino acid N-terminal region, 30 tandem repeats consisting of a 24-amino acid sequence that makes up the core of the protein, and a 103-amino acid-long C-terminal region with homology to epidermal growth factor (EGF)2 [6]
Our prior finding that BAD-1 binding to yeast cell surfaces depends on the concentration of calcium [10], taken together with the fact that BAD-1 consists of 30 EF-hand-like motifs strung together, raised the possibility that BAD-1 could be a calciumbinding protein
Summary
Aspartic acid-rich repeats contiguous to an EGF module. We investigated BAD-1 binding of calcium, as well as the structural basis and functional consequences of this interaction. We demonstrate a newly appreciated function of BAD-1 involving high capacity calcium binding. The binding of calcium by BAD-1 in turn has functional consequences on the localization of BAD-1 and the ability of the fungus to survive conditions of calcium limitation
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