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J. Biol. Chem., Vol. 280, Issue 51, 42156-42163, December 23, 2005

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Calcium Binding by the Essential Virulence Factor BAD-1 of Blastomyces dermatitidis^*

T. Tristan Brandhorst, Gregory M. Gauthier, Richard A. Stein, and Bruce S. Klein¶||1

From the Departments of Pediatrics, Internal Medicine, and ^¶Medical Microbiology and Immunology and the ^||Comprehensive Cancer Center, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics, Madison, Wisconsin 53792

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.

Received for publication, July 1, 2005 , and in revised form, October 21, 2005.

^* This work was supported by funds from the United States Public Health Service (to B. S. K.), the Parker B. Francis Foundation (to T. T. B.), and the Infectious Disease Society of America (to G. M. G.). The University of Wisconsin-Madison Biophysics Instrumentation Facility, where the CD experiments were performed, was established by National Science Foundation Grant BIR9512577 and National Institutes of Health Grant RR13790. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: University of Wisconsin-Madison, 600 Highland Ave., K4/434, Madison, WI 53792. Tel.: 608-263-9217; Fax: 608-263-6210; E-mail: bsklein{at}wisc.edu.

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