Unexpected fold in the circumsporozoite protein target of malaria vaccines

Abstract

Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an "αTSR" domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

Fig. 1.

Sequence alignments and fold schematics. (A) Gene architecture of P. falciparum circumsporozoite protein. The portion of CS fused to hepatitis B surface antigen in the RTS,S vaccine is indicated by “vaccine.” (B) C-terminal portion of CS from different species. Conserved residues are colored according to chemical type. Disulfide-linked cysteines are colored according to connectivity. Dots below green (hydrophobic) columns mark residues in the hydrophobic core (blue) or exposed hydrophobic pocket (orange). Secondary structure is designated below the alignment by thick lines (helices and β-strands) colored by TSR strand direction (cyan and pink are antiparallel strand directions). Shannon sequence entropy was calculated from 42 distinct P. falciparum isolates from Freetown, Sierra Leone (27) using the Protein Variability Server (46). (C) Alignment with TSR domains from F-spondin (FSP) (9), thrombospondin-1 (TSP1) (7), and ADAMTS13 (10). Unique portions of PfCS αTSR are overlined in orange. Only sequences overlined in identical colors are structurally equivalent. Segments forming sheets or helices are shown with thickened lines. Disulfide connectivity is color coded. P. falciparum CS (3D7 strain) mature numbering is shown above the sequences. TSR consensus residues are shown below the alignment. (D) Diversity of TSR domain architectures, modified from ref. .

Fig. 2.

Structure of αTSR. (A) CS αTSR showing layer residues and conserved flap fastening residues. (B) Layers of the TSR fold. Dashed boxes indicate layers lost in the αTSR. Dashed blue lines indicate π-cation and salt bridge interactions retained in αTSR, adapted from ref. . (C) Thrombospondin-1 TSR domain 1 (7) superimposed on the αTSR domain in A, Right and separated horizontally. Trp layers are colored orange and stacking layers are labeled by residue type. (D) Hydrophobic core of the αTSR domain. Conserved hydrophobic sidechains (with the exception of I346, in which many species have arginine or lysine sidechains) and their interactions are shown. A portion of the structure is omitted for clarity. (E) Transparent surface representation of αTSR with conserved hydrophobic pocket residues and their surface representation shaded orange. (F) Fitting of the crystal structure of αTSR to the final SWAXS envelope, shown as a transparent surface. The αTSR crystal structure is shown in cartoon in green, except the N-terminal YVEF is in orange. The YVEF sequence may be flexible in solution, because it is not included in the SWAXS envelope. Arrows indicate an observed indent on the SWAXS model corresponding to the approximate location of the hydrophobic pocket in the crystal structure. (G) Scattering curve calculated from the crystal structure and excluding the YVEF sequence (black) is overlaid to the raw experimental SWAXS scattering data for Pichia αTSR (red) and HEK293T αTSR (cyan). (H) Heparin-sepharose affinity assay of Pichia αTSR (red) and CS (black). Each construct was passed over a HiTrap heparin-sepharose column at a flow rate of 0.2 mL/min in 10 mM Tris pH 7.4 buffer and washed with a sodium chloride gradient (brown) over 40 column volumes.