Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches' Broom Disease of Cacao

Abstract

The pathogenic fungi Moniliophthora perniciosa causes Witches' Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is found in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.

Figure 1

Crystal structure and packing of MpPR-1i. (a) Cartoon of monomer A colored in rainbow from blue (N-ter) to red (C-ter). (b) MpPR-1i monomers in asymmetric unit viewed along the diagonal of unit cell shows a pseudo-seven fold screw axis; each monomer is labeled along the direction of (110) to (011) as A, B, C, D, E, F, and G respectively. (c) Crystal packing presented along a cell dimension. Monomers in the top layer are colored as in Fig. 1b, while the bottom layer monomers are shown in gray. The large solvent channel formed by crystal packing is also visible. (d) Crystal packing viewed along the cell diagonal.

Figure 2

Structure similarity and intermolecular interaction of MpPR-1i. (a) Superposition of all seven monomers in asymmetric unit reveals that loop regions at termini and between longest β-sheet as the most variable parts. (b) Comparison of C-termini of monomers A and C. The insert reveals that monomer C and A have the overall opposite orientation starting from the peptide bond between Asp157, Tyr158, and Tyr159 in molecule C rotate ~90° clockwise compared to the equivalent residues in molecule A indicated by arrow. (c) Global view of intermolecular interaction of MpPR-1i. (d) Network of interactions between monomer A and B. The carbon atoms are colored as gray in molecule A and orange in molecule B. Oxygen atoms are shown as red and nitrogen atoms are presented as blue. The hydrogen bonds are shown as black dash.

Figure 3

Comparison of CAP cavity of Pry1CAP and MpPR-1i. (a) Surface diagram of Pry1CAP and (b) MpPR-1i reveal central CAP cavity and Caveolin-binding motif (CBM) containing dioxane (orange stick), ribbon diagram of equivalent view of (c) Pry1CAP and (d) MpPR-1i monomer showing CBM containing dioxane (orange stick) and CAP tetrad (stick).

Figure 4

SCP/TAPS protein motifs. Structural features of MpPR-1i and primary sequence alignment with SCP/TAPS proteins that are most structurally similar. This figure was generated with ESPript. The different secondary structure elements shown are alpha helices as large squiggles labelled (α), 3₁₀-helices as small squiggles labelled (η), beta strands as arrows (β), and beta turns (TT). Identical residues are shown in white on red background, and conserved residues in red. The locations of the cysteine residues involved in disulfide bonds are numbered in green. CAP motifs are highlighted in orange, and caveolin-binding motif is indicated in blue. The SCP/TAPS structures with pdb accension codes in parenthesis are Na-ASP-2 (1u53), SmVAL4 (4p27), PI14a (1cfe), GAPR-1 (1smb), Pry1CAP (5ete), and sGLIPR1 (PDB entry 3q2r).

Figure 5

MpPR-1i has a large lipid binding site like tablysin-15. (a) Superpositioning of MpPR-1i (gray) with tablysin-15 (blue) reveals similar sized palmitate (magenta) binding cavity. (b) Surface diagram reveals that the cavity is large enough to accommodate palmitate. (c) In vitro binding affinity of palmitate to MpPR-1i.