Structural features responsible for the biological stability of Histoplasma's virulence factor CBP

Abstract

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.

Figure 1

ESI-MS spectra of (A) calcium-free and (B) calcium-loaded CBP show that calcium can be completely removed from the protein preparation and that several different calcium-bound forms exist in the gaseous phase of electrospray ionization. Calcium-free CBP was obtained after the sample was passed over a Calcium-Sponge, whereas 10 mM CaCl₂ was added to generate the calcium-loaded CBP sample in identical buffer [10 mM ammonium acetate (pH 7.0)].

Figure 2

Selected sedimentation equilibrium data and fits for CBP. Panel A shows data fit to a single-species model, and data in panel B make up a multispeed fit to a monomer–dimer equilibrium model. Experimental conditions were as follows: sample concentration of 10 (A) or 40 μM (B), rotation speeds of 25000 rpm (A) and (● in panel B) 35000 (red), 37000 (green), and 45000 rpm (blue), and a temperature of 20 °C. The data were fit using eq 1 and the monomeric molecular mass of 7855 Da from eq 2. Residuals are shown below the respective data or fit.

Figure 3

Far-UV CD spectra of CBP: native CBP at pH 6.5 (solid black line), reduced CBP (dashed black line), denatured CBP (solid gray line), and reduced and denatured CBP (dashed gray line). The samples contained 25 μM CBP in 0.01 M KH₂PO₄ (pH 6.5) and 0.1 M KCl at 25 °C; reduced samples also contained 15 mM DTT, and denatured samples also contained 10 M urea.

Figure 4

¹H–¹⁵N HSQC spectrum of uniformly ¹⁵N-labeled CBP at a protein concentration of 1 mM in 10 mM deuterated Hepes (pH 6.5), 100 mM KCl, and 0.02% NaN₃ at 25 °C. Backbone amide correlations are labeled according to the assignments described in text. Cross-peaks are connected by lines corresponding to Gln and Asn side chain NH₂ groups.

Figure 5

Summary of secondary structure determinants collected for CBP. (A) Secondary structure determined from the consensus chemical shift index, the residue numbers, and the amino acid sequence of mature CBP. (B) Measured chemical shift indices for CBP, where positive CSI values correspond to regions of β-sheet and negative values reflect regions of α-helix. (C) NOE connectivities, which are classified by the NOE volume (strong or weak) and are reflected in the thickness of the bars. (D) Measured scalar coupling constants (³J_HNHA), with values between 2 and 6 corresponding to α-helical bond angles.

Figure 6

Summary of CBP proteolysis. Successful cleavage sites identified by ESI-MS after proteolysis are indicated above the sequence of CBP for digests with trypsin, Glu-C, Asp-N, or Lys-C indicated by white triangles, whereas missed cleavage sites are indicated by black triangles. Underlined segments represent disulfide-linked peptides identified by 50% H₂ ¹⁸O proteolysis with pepsin. The final resolved disulfide linkages are designated with brackets and numbered by cysteine positions.

Figure 7

Thermal denaturation monitored by CD of CBP at protein concentrations of 5 (■), 10 (×), 20 (+), and 50 μM (○) in 0.01 M KH₂PO₄ and 0.1 M KCl (pH 6.5). Solid lines represent the results of fitting the data to a two-state unfolding model, with residuals plotted at the top.

Figure 8

Urea-induced denaturation monitored by CD of CBP (25 μM) in 0.01 M KH₂PO₄ and 0.1 M KCl (pH 6.5) at room temperature. The solid line represents the results of fitting the data (○) to a two-state unfolding model with residuals plotted at the top.