Crystal structure of the Mp1p ligand binding domain 2 reveals its function as a fatty acid-binding protein

Abstract

Penicillium marneffei is a dimorphic, pathogenic fungus in Southeast Asia that mostly afflicts immunocompromised individuals. As the only dimorphic member of the genus, it goes through a phase transition from a mold to yeast form, which is believed to be a requisite for its pathogenicity. Mp1p, a cell wall antigenic mannoprotein existing widely in yeast, hyphae, and conidia of the fungus, plays a vital role in host immune response during infection. To understand the function of Mp1p, we have determined the x-ray crystal structure of its ligand binding domain 2 (LBD2) to 1.3 A. The structure reveals a dimer between the two molecules. The dimer interface forms a ligand binding cavity, in which electron density was observed for a palmitic acid molecule interacting with LBD2 indirectly through hydrogen bonding networks via two structural water molecules. Isothermal titration calorimetry experiments measured the ligand binding affinity (K(d)) of Mp1p at the micromolar level. Mutations of ligand-binding residues, namely S313A and S332A, resulted in a 9-fold suppression of ligand binding affinity. Analytical ultracentrifugation assays demonstrated that both LBD2 and Mp1p are mostly monomeric in vitro, no matter with or without ligand, and our dimeric crystal structure of LBD2 might be the result of crystal packing. Based on the conformation of the ligand-binding pocket in the dimer structure, a model for the closed, monomeric form of LBD2 is proposed. Further structural analysis indicated the biological importance of fatty acid binding of Mp1p for the survival and pathogenicity of the conditional pathogen.

FIGURE 1.

GC/MS determination of LBD2 ligands. Fatty acid chain lengths and saturation states are indicated.

FIGURE 2.

Overall structure of the Mp1p LBD2, structural model for full-length Mp1p, and the possible conformation changes during ligand binding. A, a ribbon diagram of the LBD2 monomer. The ribbon diagram is shown with the protein molecule colored pink and the palmitic acid ligand colored green. B, a ribbon diagram of the LBD2 homodimer. The two protein molecules are colored pink and chartreuse, respectively, and palmitic acid is colored yellow. C, a schematic for the closed form of the ligand-bound LBD2 monomer. The LBD2 molecule is colored pink and shown in ribbon representation; palmitic acid is colored green. The crystal structure of the LBD2 dimer is used as a model for the closed form conformation. D, a structural model for full-length Mp1p. The N-terminal signal peptide and C-terminal glycosylphosphatidylinositol sequence are colored purple. LBD1 and LBD2 are colored pink and green, respectively, and are shown in cartoon using the crystal structure of LBD2. The third domain is colored blue. The model is a schematic of Mp1p structure, not an interpretation of the real domain interactions in full-length protein. E, a schematic diagram for the possible conformation of Mp1p LBD1 and LBD2 domains bound with palmitic acid. The two LBD domains are colored pink and green, respectively, and palmitic acid molecules are colored yellow.

FIGURE 3.

AUC results for WT Mp1p and WT LBD2, delipidated or with PA at different concentrations. The AUC results are represented as a c(M) distribution model. A, the blue curve refers to WT Mp1p. B, the green curve stands for Mp1p after delipidation process. C and D, the orange (C) and pink curves (D) refer to the samples of delipidated Mp1p added with PA at molar ratio 1:2 and WT Mp1p with PA at molar ratio 1:8, respectively. E, the red curve refers to WT LBD2. F, the purple curve represents LBD2 after delipidation. G and H, the brown (G) and gray curves (H) refers to the samples of delipidated LBD2 added with PA at molar ratio 1:1 and WT LBD2 with PA at molar ratio 1:1.5, respectively.

FIGURE 4.

A, surface representation of Mp1p LBD2 in complex with palmitic acid. Palmitic acid is shown in ball-and-stick representation and colored by atom type (carbon in green and oxygen in red.) Water molecules are shown as purple spheres. Residues at the ligand binding site are labeled; the surface is colored pink for hydrophobic residues and blue for the rest. Surface residues outside the ligand binding site are colored red. Residues involved in hydrogen bonding networks are shown in stick representation. The dashed lines show the hydrogen bonds between the residues and water molecules. B, palmitic acid overlaid by a 2F_o − F_c electron density map contoured at 1.0 σ. Palmitic acid is shown in stick representation and colored by atom type (carbon in green and oxygen in red). The density map comes from the original single-wavelength anomalous diffraction phased map generated by RESOLVE. C, stereo view of the ligand binding site of Mp1p LBD2. Palmitic acid is colored by atom type (carbon in green and oxygen in red). Water molecules are shown as purple spheres. Residues involved in hydrogen bonding networks are shown in ball-and-stick representation and colored by atom type (carbon in white, oxygen in red, and nitrogen in blue). Residues forming hydrophobic interactions with palmitic acid are colored by atom types (carbon in pink and blue for subunits A and B, respectively; oxygen in red; and nitrogen in blue). The helixes are shown as a ribbon diagram and colored red and blue for subunits A and B, respectively. The dashed lines show the hydrogen bonds between the residues and water molecules.

FIGURE 5.

Titration of Mp1p with PA. Binding isotherms were derived from the raw data shown in the insets, with 100 μm Mp1p at 25 °C.

FIGURE 6.

Sequence alignment of Mp1p (P. marneffei) with homologous proteins. From top to bottom, the sequences are: LBD2 of Mp1p from P. marneffei; LBD1 of Mp1p from P. marneffei; NFMP2 from N. fischeri; AFMP1 from A. fumigatus; AFMP2 from A. fumigatus. The last three aligned sequences are the putative LBD domains from the three different proteins. The secondary structure of Mp1p (P. marneffei) is indicated at the top of the alignment. Strictly conserved residues among the aligned sequences are indicated in red, and the most conserved residues are shaded in red. Residues involved in hydrogen bond formation or hydrophobic interaction with palmitic acid are labeled with blue and green triangles, respectively. The LBD2 residues are numbered above the sequence.

FIGURE 7.

Possible mode of action of P. marneffei Mp1p in fatty acid host-pathogen trafficking. LBD1 and LBD2 are colored pink and green, respectively. The four-helix bundles are represented by larger ovals, whereas the two-helix bundles are represented by smaller ovals. The fatty acids are indicated as small yellow ovals.