Structure of the Pf12 and Pf41 heterodimeric complex of Plasmodium falciparum 6-cysteine proteins

Abstract

During the different stages of the Plasmodium life cycle, surface-associated proteins establish key interactions with the host and play critical roles in parasite survival. The 6-cysteine (6-cys) protein family is one of the most abundant surface antigens and expressed throughout the Plasmodium falciparum life cycle. This protein family is conserved across Plasmodium species and plays critical roles in parasite transmission, evasion of the host immune response and host cell invasion. Several 6-cys proteins are present on the parasite surface as hetero-complexes but it is not known how two 6-cys proteins interact together. Here, we present a crystal structure of Pf12 bound to Pf41 at 2.85 Å resolution, two P. falciparum proteins usually found on the parasite surface of late schizonts and merozoites. Our structure revealed two critical interfaces required for complex formation with important implications on how different 6-cysteine proteins may interact with each other. Using structure-function analyses, we identified important residues for Pf12-Pf41 complex formation. In addition, we generated 16 nanobodies against Pf12 and Pf41 and showed that several Pf12-specific nanobodies inhibit Pf12-Pf41 complex formation. Using X-ray crystallography, we were able to describe the structural mechanism of an inhibitory nanobody in blocking Pf12-Pf41 complex formation. Future studies using these inhibitory nanobodies will be useful to determine the functional role of these two 6-cys proteins in malaria parasites.

Keywords: 6-cysteine proteins; Nanobodies; Plasmodium falciparum; X-ray crystallography; blood stages; malaria.

Figure 1.

Crystal structure of the heterodimeric complex of Pf12-Pf41. (A) Schematic diagram of Pf12 and Pf41 (left, right respectively). SP, signal peptide; GPI, GPI-anchor; D1, N-terminal 6-cys domain; D2, C-terminal 6-cys domain; ID, inserted-domain like region. Residue numbers are indicated. (B) Structure of the Pf12-Pf41 complex in two orthogonal views as surface representation (left) and ribbon representation (middle, right). The two 6-cys domains, D1 and D2, are shown in light and dark purple for Pf12 and in light and dark green for Pf41 respectively. Within Pf41, the inserted domain-like region (ID) which is located between the last two β-strands of D1 is coloured yellow. A dashed arrow indicates the linker region of 17 residues between the C-terminus of Pf12 D1D2 and the predicted GPI-anchor attachment site (S321) which were not included in the crystallization construct. (C) The Pf12-Pf41interface. Pf12 residues that interact with the ID of Pf41 are shown in yellow; residues that interact with the C-terminal D2 domain of Pf41 are shown in dark green. Pf41 residues that are in contact with Pf12 D1 and D2 are shown in light and dark purple, respectively. Footprints for binding sites are defined by contacting residues within 5.0 Å. (D) Interactions between the Pf12 D1-D2 interdomain region and the Pf41 D2 domain in two different orientations (top panels, left and right). Interactions between the Pf12 D2 domain and the Pf41 ID shown in two different views (bottom panels, left and right). (E) Structural overlay of Pf12 D1D2 with the published unbound Pf12 crystal structure, PDB ID 2YMO, aligned based on the D2 domain (left). Structural overlay of Pf41 D1D2 with the published unbound Pf41 crystal structure, PDB ID 4YS4 (right).

Figure 2.

Structure-function analysis of Pf12 and Pf41 residues involved in complex formation. (A) Schematic diagram of recombinant proteins Pf12 D1D2 and Pf41 D1D2 with the mutated interfacing residues indicated with arrows. (B) Effect of mutations on Pf12–Pf41 complex formation. BLI-measurements between Pf12 and Pf41 wildtype and mutant proteins. Relative maximum response of each mutant at 1000 nM, 500 nM, and 250 nM concentration (represented by the dots) compared to wildtype protein maximum response is shown. Wildtype–wildtype binding was assigned to 100%. Dotted lines indicate loss of binding (<20% binding), strong loss of binding (21%–45%), intermediate loss of binding (46%–69%) and similar or no loss of binding (>70%) compared to wildtype. Bars represent the mean ± standard deviation (SD) for the three concentrations.

Figure 3.

Pf12 and Pf41 specific nanobodies. (A) Sequence alignment of 16 nanobodies with framework regions (FR) and complementary determining regions (CDR) indicated according to the international ImMunoGeneTics information system (IMGT). Residues that represent less than 60% similarity to the consensus sequence are shown in black. (B) Coomassie-stained SDS-PAGE gels of purified nanobodies under reducing conditions. Molecular weight marker (M) in kDa is shown on the left. (C) Domain mapping of Pf12- and Pf41-specific nanobodies using BLI. Pf12 D1 and Pf12 D2 were examined for binding to anti-Pf12 nanobodies (left). Pf41 D2 and Pf41 ΔID, were tested for binding to anti-Pf41 nanobodies (right). Summary of nanobodies that recognise the respective protein domains are shown on the right of each schematic. (D) Iso-affinity plot showing the association rate constants (ka) vs dissociation rate constants (kd) of Pf12 or Pf41 binding to their respective nanobodies. Diagonal lines indicate equilibrium dissociation rate constants (K_D). The area of the plot highlighted in grey indicates the detection limit of the instrument.

Figure 4.

Pf12 specific nanobodies can inhibit Pf12-Pf41 complex formation. (A) Anti-Pf12 nanobodies inhibit Pf12-Pf41 complex formation in a FRET-based assay. The FRET signal is relative ‘no nanobody’ control. B9 is a nanobody specific for 6-cys protein Pf12p and does not recognise Pf12 or Pf41 (Dietrich et al. 2021). Nanobodies specific for Pf12 and Pf41 are indicated. Error bars represent standard error of the mean calculated from three independent measurements. Open circles represent the individual replicates. (B) Crystal structure of inhibitory Nb G7 in complex with Pf12 shown in two orthogonal views. All three CDR loops of Nb G7 (backbone, grey; CDR loops, blue) binds to the D2 domain of Pf12 (dark purple). (C) The Nb G7 epitope overlaps with the Pf41 ID binding site on Pf12. Pf12 is shown in surface representation (purple). The footprint of the three CDR loops of G7 are coloured in three different shades of blue and the footprint of Pf41 is indicated with a yellow outline. (D) Steric hindrance of the Pf12 D2 binding site. Secondary-structure matching (SSM) of Pf12 bound to Pf41 and Pf12 bound to Nb G7 based on the Pf12 D2 domains. Pf12 of Pf12-Nb G7 is shown in surface representation. Nb G7 (grey) and the insertion domain ID of Pf41 (yellow, shown in ribbon representation) clash as both proteins occupy a similar space around the Pf12 D2 domain.