The structure of Plasmodium falciparum 3D7_0606800 reveals a bi-lobed architecture that supports re-annotation as a Venus Flytrap protein

Abstract

Plasmodium falciparum, the causative agent of malaria, employs a diverse array of surface displayed proteins to promote dissemination and establish infection in the human host. Of these, Pf3D7_0606800 is highly immunogenic and has been designated a potential top 10 candidate for inclusion in a multicomponent malarial vaccine. The role of Pf3D7_0606800 in parasite biology, however, is unknown and its characterization has been complicated by a lack of sequence identity with proteins of known structure or function. Towards elucidating Pf3D7_0606800 function, we determined its structure to a resolution of 2.35 Å using selenium single wavelength anomalous dispersion. A bi-lobed architecture displays the core structural hallmarks of Venus Flytrap (VFT) proteins prompting us to re-annotate Pf3D7_0606800 as PfVFT1. Structural analysis further revealed an extended inter-lobe groove that, when interrogated by molecular docking, appears well suited to bind peptide-based ligands. Collectively, our structural characterization of the highly antigenic P. falciparum surface protein PfVFT1 provides intriguing functional insight and establishes a structural template that could prove valuable for malaria vaccine engineering studies.

Keywords: Plasmodium falciparum; X-ray crystallography; bi-lobed architecture; conformational flexibility; venus flytrap domain.

Figure 1

Structural characterization of Pf3D7_0606800 reveals a bi‐lobed domain architecture. A: Top: Schematic of the Pf3D7_0606800 protein sequence. Bottom: SEC profile of native (blue line) and SeMet (black line) Pf3D7_0606800 produced in insect cells. Inset, Coomassie‐stained SDS‐PAGE gel of Native and SeMet Pf3D7_0606800 (expected molecular weight: 32 kDa). B: Cartoon representation of orthogonal views of Pf3D7_0606800 structure. C: Surface representation of Pf3D7_0606800 highlighting the central cleft (blue) with the hydrophobic (orange dots) and aromatic (yellow dots) residues. D: Orthogonal views of Pf3D7_0606800 electrostatic surface representation. An interactive view is available in the electronic version of the article.

Figure 2

Pf3D7_0606800 adopts a Venus Flytrap (VFT) domain fold and is re‐annotated as PfVFT1. A: Secondary structure elements that are conserved (gray) and that diverge (purple, green, and blue) between PfVFT1 and StABC (PDB ID 3HV1). Topology diagram of PfVFT1 highlighting unique N‐terminus (blue components prior to β1, deep purple) and C‐terminus (green). Starburst and dotted connector indicate a disulfide bond. Gray dotted indicates lobe 2 helix (yellow outline) that is common to most VFTs, but absent in PfVFT1. B: A phylogenetic tree assembled using structure‐based (RMSD) alignments of PfVFT1 with two representative members for every cluster, obtained from previous evolutionary analysis on VFTs14 using PDBefold.15, 16 For simplicity, the recently identified Cluster G14 was not included. Escherichia coli (Ec); Homo sapiens (Hs); Yersinia pesits (Ys); Halomonas elongate (He); Ruegeria pomeroyi (Ru); Streptococcus thermophiles (St); Synechocystis spp. (Ss). EcFhuD‐1PSZ; EcFnbp‐2CHU; EcLBP‐1USK; EcLIV‐1Z16; EcMBP‐4MBP; HsMBP‐2ZVK; EcPBP‐1IXH, YsPBP‐2Z22; HeSBP‐2VPO; RuSBP‐3FXB; StABC‐3HV1; SsCmpa‐2I48; SsNrta‐2G29. An interactive view is available in the electronic version of the article.

Figure 3

Peptide coordination in the inter‐lobe cleft. A: Left: Plasmodium spp. VFT1 polypeptide sequences were aligned using Clustal Omega21 and a phylogenetic tree rooted to the divergent PfVFT1 homolog in T. gondii was generated using MEGA22 with 500 bootstrap replicates (See methods for accession numbers). Right: mapping of conserved (burgundy) and variable (cyan) residues of PfVFT1 homologs onto the PfVFT1 surface using ConSurf23; conservation of cleft region indicated by black border. B: Interactions between the N‐terminal tail of Chain A and the inter‐lobe cleft of Chain B. Inset: residues from Chains B and A are labeled green and yellow, respectively. C: Left: Weblogo of the consensus sequence of the 500 best‐scored tripeptides and respective docking result to PfVFT1. Right: Energy minimized docking pose of the consensus peptide.