Nanobody generation and structural characterization of Plasmodium falciparum 6-cysteine protein Pf12p

Abstract

Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are β-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 Å resolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated 10 distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.

Keywords: 6-cysteine proteins; malaria; nanobody.

Figure 1.. Pf12p-specific nanobodies.

(A) Sequence alignment of 10 nanobodies with framework regions (FR) and complementary determining regions (CDR) indicated according to the international ImMunoGeneTics information system (IMGT). Black residues represent less than 60% similarity to the consensus sequence. (B) Coomassie-stained SDS–PAGE gel of purified Pf12p-specific nanobodies under reducing conditions. Molecular mass marker (M) in kDa is shown on the left-hand side. (C) Detection of Pf12p by nanobodies using ELISA. Anti-Pf12p nanobodies, anti-Pf12 nanobody D12 and anti-Pf41 nanobody A4 were added to microtiter wells coated with Pf12p, Pf12 and Pf41. Bound nanobodies were detected with anti-His antibody followed by HRP-conjugated secondary antibody. Error bars represent standard deviation of the mean. (D) Detection of Pf12p by nanobodies by Western blotting. Reduced (R) and non-reduced (NR) Pf12p protein was separated by SDS–PAGE and probed with the respective nanobodies and detected using an HRP-conjugated goat anti-llama IgG. Molecular mass marker in kDa is shown on the left-hand side. (E) Iso-affinity plot showing the dissociation rate constants (k_d) and association rate constants (k_a) of Pf12p nanobodies as measured by BLI. Symbols that fall on the same diagonal lines have the same equilibrium dissociation rate constants (K_D) indicated on the top and right sides of the plot.

Figure 2.. Domain mapping and epitope competition of Pf12p-specific nanobodies.

(A) Domain organization of full-length Pf12p (upper) and recombinant fragments of Pf12p D1D2 (middle) and Pf12p D2 (lower). SP, signal peptide; GPI, predicted GPI-anchor sequence; His₈-TEV, N-terminal His₈-tag followed by a TEV-cleavage site. Lines and numbers show cysteine bonds. (B) Domain mapping of Pf12p-specific nanobodies using ELISA. Anti-Pf12p nanobodies were added to microtiter wells coated with Pf12p D1D2 and Pf12p D2. Bound nanobodies were detected with anti-His antibody followed by HRP-conjugated secondary antibody. Error bars represent standard deviation of the mean. (C) Epitope competition experiments by BLI using immobilized nanobodies indicated on the left column incubated with nanobodies indicated on the top row pre-incubated with Pf12p using a 10 : 1 molar ratio. Binding of Pf12p premixed with nanobody was calculated relative to Pf12p binding alone, which was assigned to 100%. A blue to red gradient shows antibodies with the highest levels of competition in blue and the lowest in red.

Figure 3.. Crystal structure of Pf12p and comparison with structures of other 6-cys protein family members.

(A) The Pf12p structure (PDB ID 7KJ7) is shown in two orthogonal views. The N- and C-termini and disulfide bonds are labelled. Dashed lines indicate regions which do not have defined electron density. The β-sheets A and B of the β-sandwich of each domain are coloured in orange and red, respectively. (B) Schematic diagram of selected 6-cys proteins (not to scale). Predicted 6-cys domains are in white and labelled sequentially. The recombinant fragments used in published structural studies are coloured. SP, signal peptide; GPI, GPI-anchor. Residue numbers are indicated on top. (C) Structural alignment of Pf12 (PDB ID 2YMO) and Pf41 (PDB ID 4YS4) with Pf12p based on the D2 domain. (D) Superimposition of Pf12p and Pf12 D1 domains (upper) and the D2 domains (lower). (E) Superimposition of Pf12p and Pf41 D1 domains (upper) and the D2 domains (lower). (F) Superimposition of the D1 domains of Pf12p and Pfs230 (D1M construct) (PDB ID 6OHG). (G) Superimposition of the D2 domain of Pf12p with the D3 domain (6C construct) of Pfs48/45 (PDB ID 6E63).

Figure 4.. Pf12p does not interact with Pf41.

(A) Sequence alignment of Pf12p and Pf12 with |, : and . indicating identical amino acids, strongly similar residues and residues of weak similarity, respectively. (B) The Pf12p structure is shown in two different views with residues that align perfectly with Pf12 residues in in the sequence alignment coloured red, residues with strong or weak similarity coloured orange and yellow, respectively. (C) BLI-binding experiment with immobilized Pf41 and Pf12 in solution. Representative binding curves of five different Pf12 concentrations are plotted and fitted to a 1 : 1 binding model. (D) BLI-binding experiment with immobilized Pf41 and Pf12p in solution. Six different Pf12p concentrations ranging from 16 to 500 nM were tested, but no binding could be detected. (E) SEC analyses show that recombinant Pf12 and Pf41 form a heterodimer. The Pf12–Pf41 complex elutes at a retention volume corresponding to higher molecular mass compared with the individual proteins on SEC. Excess of Pf12 runs as a shoulder of the complex-peak at the expected retention volume. (F) SEC analyses show that recombinant Pf12p does not form a stable complex with Pf41. The mix of protein elutes at a retention volume between the peak maxima of the individual proteins. Retention volume of molecular mass marker proteins and their corresponding size are indicated. For panels E and F, the bottom part presents the SDS–PAGE gels for fractions at the same retention volume of each run.

Figure 5.. Crystal structures of Pf12p in complex with nanobody B9 and D9, respectively.

(A) Structure of Pf12p bound to nanobody B9. (B) Structure of Pf12p bound to nanobody D9. For panel (A) and (B), the complementary determining regions (CDR) are coloured in light blue (CDR1), blue (CDR2), and dark blue (CDR3). (C) Footprint of nanobody B9 on Pf12p D1 and D2 domains. (D) Footprint of nanobody D9 on Pf12p D1 and D2 domains. The Pf12p D1 and D2 domains are shown in surface representation in light and dark grey, respectively. The footprint of CDR loops is coloured as described in panel (A) and (B). Coloured Pf12p residues represent those that contact the nanobodies within a distance cutoff of 5 Å. The interaction surface area is indicated. (E) Structural alignment of five Pf12p molecules derived from the asymmetric units of the three structures Pf12p (PDB ID 7KJ7), Pf12p bound to nanobody B9 (PDB ID 7KJH) and Pf12p bound to nanobody D9 (PDB ID 7KJI).