Structural features responsible for the biological stability of Histoplasma's virulence factor CBP.
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 Ca (2+) 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 alpha-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 alpha-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.
Beck
Moriah R
MR
Department of Molecular Microbiology, Washington University, St. Louis, Missouri 63110, USA.
DeKoster
Gregory T
GT
Hambly
David M
DM
Gross
Michael L
ML
Cistola
David P
DP
Goldman
William E
WE
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006658:Histoplasma / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D014461:Ultracentrifugation; D037521:Virulence Factors / Q000737:chemistry* / Q000378:metabolism
D002135:Calcium-Binding Proteins; C416373:Cbp1 protein, histoplasma; D004220:Disulfides; D005656:Fungal Proteins; D010455:Peptides; D037521:Virulence Factors; D002118:Calcium