The hypothetical protein P47 of Clostridium botulinum E1 strain Beluga has a structural topology similar to bactericidal/permeability-increasing protein

Abstract

Botulinum neurotoxins (BoNTs) are causative agents of the life-threatening disease botulism. They are naturally produced by species of the bacteria Clostridium botulinum as stable and non-covalent complexes, in which the BoNT molecule is assembled with several auxiliary non-toxic proteins. Some BoNT serotypes, represented by the well-studied BoNT serotype A (BoNT/A), are produced by Clostridium strains that carry the ha gene cluster, which encodes four neurotoxin-associated proteins (NTNHA, HA17, HA33, and HA70) that play an important role to deliver and protect BoNTs in the gastrointestinal tract during oral intoxication. In contrast, BoNT/E- and BoNT/F-producing strains carry a distinct gene cluster that encodes five proteins (NTNHA, P47, OrfX1, OrfX2, and OrfX3, termed the orfX cluster). The structures and functions of these proteins remain largely unknown. Here, we report the crystal structure of P47 resolved at 2.8 Å resolution. Surprisingly, P47 displays a structural topology that is similar to bactericidal/permeability-increasing (BPI) like proteins, which were previously identified only in eukaryotes. The similarity of a hydrophobic cleft of P47 with the phospholipid-binding groove of BPI suggests that P47 might be involved in lipid association to exert its function. Consistently, P47 associates and induces aggregation of asolectin-containing liposomes in a protein- and lipid-concentration dependent manner. These findings laid the foundation for future structural and functional studies of the potential roles of P47 and OrfX proteins in facilitating oral intoxication of BoNTs.

Keywords: Bactericidal/permeability-increasing protein; Botulinum neurotoxin; Crystal structure; Lipid binding; P47; Toxin complex; orfX gene cluster.

Fig. 1. Biochemical characterization of P47

(A) SEC analysis of the recombinant P47. P47 is homogeneously monomeric with purity higher than 95 % judged by SDS-PAGE (Boxed). (B) Thermo stability of P47 as a function of pH. The data are presented as mean ± S.D., n = 3.

Fig. 2. Crystal structure of P47

(A) P47 has two major domains (green and blue) that are connected by a flexible loop (magenta). Three anti-parallel strands from each domain interact to form a six-stranded β-sheet in the middle of P47 (highlighted in a box). (B) Molecular surface of P47 is shown and colored according to the electrostatic potential (±3kT/e, red: negative; blue: positive) that was calculated using APBS (Baker et al., 2001). P47N is more electronegative than P47C. (C) Structural comparison of P47N and P47C. The secondary structures are labeled to highlight the similar topology of these two domains. The two β-strands that are unique to P47N and P47C are labeled as β6e and β1e, respectively. (D) Sequence alignment of P47N and P47C performed by PROMALS3D (Pei et al., 2008) and displayed using ESPript 3.0 (Robert & Gouet, 2014). Their secondary structures are placed on the top and the bottom, respectively. Identical residues are white-colored and red-boxed. Conserved residues are red-colored and blue-framed.

Fig. 3. Structural comparison of P47 with BPI reveals a potential lipid-binding pocket

(A) The P47 domains are colored the same as in Fig. 2. The N- and C- terminal domains of BPI (PDB code: 1BP1) are colored pink and yellow respectively. The phospholipid is represented as purple spheres. (B) Structural alignment of P47C with the N-terminal domain of BPI (DaliLite v.3). β1-β2 hairpin of P47, β2-β3 and β4- β5 hairpins of BPI and P47 are marked. Positively charged residues on BPI that are crucial for LPS binding are shown as spheres. (C) Hydrophobic residues in BPI that interact with the bound phospholipid are shown as sticks (left panel). The corresponding region in P47 is shown in the right panel, where six phenylalanine residues occupy the potential lipid-binding pocket. (D) P47 induces aggregation of asolectin-liposome. P47 at indicated concentration was added to 0.2 mM liposomes, and the changes of solution turbidity were measured. (E) Protein-liposome aggregates were analyzed by SDS-PAGE. 400 nM of P47 was incubated with liposomes at indicated concentration and the aggregates were isolated by centrifugation. The supernatant (S) and the pellet fractions (P) were separated and analyzed by SDS-PAGE.