Structural Features Responsible for the Biological Stability of Histoplasma’s Virulence Factor CBP

• Published in: Biochemistry (Easton) 2008-04, Vol.47 (15), p.4427-4438
• Main Authors: Beck, Moriah R, DeKoster, Gregory T, Hambly, David M, Gross, Michael L, Cistola, David P, Goldman, William E
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Calcium - metabolism; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - metabolism; Circular Dichroism; Dimerization; Disulfides - chemistry; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Histoplasma - pathogenicity; Histoplasma capsulatum; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides - chemistry; Protein Denaturation; Protein Structure, Secondary; Ultracentrifugation; Virulence Factors - chemistry; Virulence Factors - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701495v

The virulence factor CBP is the most abundant protein secreted by Histoplasma capsulatum, a pathogenic fungus that causes histoplasmosis. Although the biochemical function and pathogenic mechanism of CBP are unknown, quantitative Ca2+ binding measurements indicate that CBP has a strong affinity for calcium (K D = 6.45 ± 0.4 nM). However, no change in structure was observed upon binding of calcium, prompting a more thorough investigation of the molecular properties of CBP with respect to self-association, secondary structure, and stability. Over a wide range of pH values and salt concentrations, CBP exists predominantly as a stable, noncovalent homodimer in both its calcium-free and -bound states. Solution-state NMR and circular dichroism (CD) measurements indicated that the protein is largely α-helical, and its secondary structure content changes little over the range of pH values encountered physiologically. ESI-MS revealed that the six cysteine residues of CBP are involved in three intramolecular disulfide bonds that help maintain a highly protease resistant structure. Thermally and chemically induced denaturation studies indicated that unfolding of disulfide-intact CBP is reversible and provided quantitative measurements of protein stability. This disulfide-linked, protease resistant, homodimeric α-helical structure of CBP is likely to be advantageous for a virulence factor that must survive the harsh environment within the phagolysosomes of host macrophages.