Unexpected fold in the circumsporozoite protein target of malaria vaccines Michael B. Doud, Adem C. Koksal, Li-Zhi Mi, Gaojie Song, Chafen Lu, and Timothy A. Springer1 Immune Disease Institute, Children’s Hospital Boston and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Timothy A. Springer, April 4, 2012 (sent for review March 19, 2012) Circumsporozoite (CS) protein is the major surface component of Plas- The CS structures reveal a unique “αTSR” domain that is modium falciparum sporozoites and is essential for host cell invasion. related to TSR domains yet has unique features (Figs. 1 C and D A vaccine containing tandem repeats, region III, and thrombospondin and 2). Region III forms an integral part of the fold as a unique type-I repeat (TSR) of CS is efficacious in phase III trials but gives only amphipathic α-helix running orthogonally to the classic three- a 35% reduction in severe malaria in the first year postimmunization. stranded TSR homology region. This α1-helix, together with We solved crystal structures showing that region III and TSR fold into a “CS flap” (Fig. 1 C and D), decorates one face of the TSR asingleunit,an“αTSR” domain. The αTSR domain possesses a hydro- β-sheet (Fig. 2A). By packing against the β-sheet, the region III phobic pocket and core, missing in TSR domains. CS binds heparin, but α-helix gives the αTSR domain a hydrophobic core lacking in αTSR does not. Interestingly, polymorphic T-cell epitopes map to spe- TSR domains. A short linker containing a conserved hydro- cialized αTSR regions. The N and C termini are unexpectedly close, phobic residue connects the helix to TSR strand 1 (Fig. 1B). providing clues for sporozoite sheath organization. Elucidation of TSR domains contain three strands; strand 1 is not a β-strand, a unique structure of a domain within CS enables rational design of yet strands 1, 2, and 3 are positioned alongside one another as if in next-generation subunit vaccines and functional and medicinal chem- an antiparallel β-sheet (Figs. 1D and 2C)(7–10). TSR domains ical investigation of the conserved hydrophobic pocket. are termed a layered fold because they are stabilized by eight layers of stacking residues that form one face of the sheet (Fig. 2 B C α uring a blood meal, Plasmodium sporozoites are transmitted and ). In the TSR domain, this stack is shortened to four layers, from an infected mosquito to a vertebrate host. Sporozoites and they form the side of the sheet opposite the hydrophobic core D (Fig. 2 A and B). Thus, the (Wxx)WxxW motif in strand 1 of TSR are uniformly covered with circumsporozoite protein (CS), a highly C α expressed and immunodominant surface protein antigen necessary domains (Fig. 1 ) is truncated in the CS TSR domain to the for sporozoite development and host cell targeting (1, 2). Gliding third W of this motif, Trp-331. However, the layers of basic res- idues on either side of this Trp seen in other TSRs are preserved motility enables sporozoites to move directly through host cells to B reach the vasculature and later to penetrate the liver. During (Fig. 2 ); the Trp-331 sidechain in strand 1 is sandwiched be- gliding, CS moves from the anterior to the posterior end of the tween Arg-345 in strand 2 and Lys-367 in strand 3, which form π-cation bonds to knit the three strands together. Two conserved sporozoite, where it is shed and deposited in a trail (3). CS is fi composed of an N-terminal domain, a conserved pentapeptide disul des further connect strands 1 and 3, and connect the turn protease cleavage site termed region I, a repeat region, a short between strands 1 and 2 to the penultimate C-terminal residue (Figs. 1 C and D and 2A). Strands 2 and 3 in TSR domains are conserved sequence termed region III, and a C-terminal region α fl with sequence homology to the thrombospondin type-1 repeat su- halved in length in TSR by the CS ap; however, the portion forming the β-sheet is conserved (Fig. 1 C and D). perfamily (TSR) (Fig. 1A). The C terminus of the CS TSR contains Surprisingly, αTSR contains a hydrophobic pocket that is a glycosylphosphatidylinositol (GPI) anchor attachment site respon- contiguous with the hydrophobic core. The core is formed by α1- sible for the association of CS with the sporozoite plasma mem- helix residues Ile-316, Tyr-319, and Ile-323 and strand 2 residues brane (4). The RTS,S malaria vaccine currently in phase III trials is Ile-342 and Val-344 on the face opposite that bearing layer composed of a C-terminal CS fragment comprising a portion of the residues (Fig. 2D). It is extended on one side by residues Pro-311 repeats, region III, and TSR (Fig. 1A) fused to hepatitis B surface and Ile-368, which interact with one another and are invariant or antigen (5, 6). In the absence of a 3D structure, there is currently no conserved as hydrophobic, respectively. The other side of the structural basis for vaccine improvement. hydrophobic core extends into an exposed hydrophobic pocket Results formed by the α1-helix residue Leu-320, loop residue Leu-327, flap residues Leu-358 and Tyr-360, and β3-strand residue Ile-364 Region III and TSR are distinguished from the more N-terminal (Fig. 2E). Although of unknown function, the importance of this sequence containing the linker to the repeat region by their hydrophobic pocket is emphasized by the invariantly hydropho- high sequence conservation across Plasmodium species (Fig. 1B). bic character of the residues forming its surface among species Therefore, we chose a CS fragment containing both region III of Plasmodium parasites that infect a range of hosts including and TSR as the unit for crystallization (marked above Fig. 1B). humans, nonhuman primates, rodents, and birds (gold circles This fragment contains at its C terminus the small residue that is below alignment; Fig. 1B). On the opposite side of the β-sheet, predicted on the sporozoite surface to be linked to the GPI an- an invariant salt bridge between Lys-353 and Asp-363 fastens the chor (Fig. 1B). The fragment was expressed with a signal peptide sequence and a C-terminal His tag as a protein secreted into the culture medium from the yeast Pichia pastoris or from mammalian Pichia Author contributions: M.B.D., A.C.K., L.-Z.M., G.S., C.L., and T.A.S. designed research; M.B.D., cells. The protein additionally contained a 4-residue N- A.C.K., L.-Z.M., G.S., C.L., and T.A.S. performed research; M.B.D., A.C.K., L.-Z.M., G.S., C.L., terminal Tyr-Val-Glu-Phe (YVEF) sequence derived from a re- and T.A.S. analyzed data; and M.B.D., A.C.K., and T.A.S. wrote the paper. striction enzyme cloning site. Proteins were purified by His-tag The authors declare no conflict of interest. fi fi fi ∼ af nity and gel ltration, with nal yields of 15 and 2 mg/L of Data deposition: The atomic coordinates reported in this paper have been deposited in culture medium from Pichia and HEK293T cells, respectively. the Protein Data Bank, www.pdb.org (PDB ID codes 3VDJ, 3VDK, and 3VDL). Protein from Pichia crystallized in two different forms, and their 1To whom correspondence should be addressed. E-mail: [email protected]. structures were solved using anomalous dispersion to 1.7 and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. IMMUNOLOGY 2.04 Å (Fig. 2 and Table 1). 1073/pnas.1205737109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1205737109 PNAS | May 15, 2012 | vol. 109 | no. 20 | 7817–7822 Downloaded by guest on October 1, 2021 A B C D 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. 7. flap at the apex of the domain where the aliphatic portion of the residues clustered around region I (16, 17) (Fig. 1A). In verte- Lys sidechain caps the hydrophobic core (Fig. 2A). brate hosts, cleavage in region I unmasks the αTSR domain and CS binds heparan sulfate proteoglycans (HSPGs) on hep- is thought to permit it to play a role in liver cell invasion (19). atocytes (11–13). The highly sulfated HSPGs of hepatocytes are As sometimes seen with short tags that are appended to pro- proposed to mediate targeting of sporozoites to the liver and teins used in crystallization, the N-terminal YVEF peptide en- trigger sporozoite cell invasion (14, 15).