Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion

Abstract

Protein-protein interactions (IPP) play an essential role in practically all biological processes, including those related to microorganism invasion of their host cells. It has been found that a broad repertoire of receptor-ligand interactions takes place in the binding interphase with host cells in malaria, these being vital interactions for successful parasite invasion. Several trials have been conducted for elucidating the molecular interface of interactions between some Plasmodium falciparum and Plasmodium vivax antigens with receptors on erythrocytes and/or reticulocytes. Structural information concerning these complexes is available; however, deeper analysis is required for correlating structural, functional (binding, invasion, and inhibition), and polymorphism data for elucidating new interaction hotspots to which malaria control methods can be directed. This review describes and discusses recent structural and functional details regarding three relevant interactions during erythrocyte invasion: Duffy-binding protein 1 (DBP1)-Duffy antigen receptor for chemokines (DARC); reticulocyte-binding protein homolog 5 (PfRh5)-basigin, and erythrocyte binding antigen 175 (EBA175)-glycophorin A (GPA).

Keywords: Malaria; Plasmodium; receptor-ligand structure; structure activity relationship.

Figure 1

Plasmodium life cycle and erythrocyte invasion interactions. (A). The Plasmodium life-cycle has two phases in its hosts: the asexual phase in humans (vertebrates) and the sexual phase in Anopheles mosquitos (invertebrate). Female Anopheles mosquitos’ bites transmit the parasite in its sporozoite form into host dermis [19]. The inoculated sporozoites migrate through the bloodstream until reaching, invading, and developing within the hepatocytes. Some sporozoites may remain in a latent state within the hepatocytes (hypnozoites) during Plasmodium vivax invasion while others become transformed into a new parasite form called merozoite [34]. Once merozoites are released into circulation, they invade new red blood cells (RBC) and some become gametocytes which can be ingested by other mosquitos during a new bite. The parasite begins its sexual cycle in a mosquito to give rise to new sporozoites that will be transmitted to humans thereby starting the asexual cycle again in a vertebrate host. (B). Molecular events during Plasmodium merozoite invasion of RBC. Initial contact with target cells, merozoite apical pole reorientation to ensure direct contact with host cell membrane and establishing specific high-affinity interactions, tight junction formation acting as an anchor, and creating the parasitophorous vacuole, following parasite gliding motility towards target cells where they replicate, producing 30–50 new merozoites and subsequently following their cycle to invade other RBC [7,35]. M1/19, M1/30, M1/33, M1/83: merozoite surface protein 1–19 kDa, 30 kDa, 33 kDa and 83 kDa fragments, respectively; M6/36: merozoite surface protein 6–36 kDa fragment; MSPDBL: merozoite surface protein Duffy binding-like; MSP7: merozoite surface protein 7; ETRAMP: early transcribed membrane protein; TRAg36.6, TRAg38, TRAg40, TRAg69.4 and TRAg74: Plasmodium tryptophan-rich proteins 36.6, -38, -40, -69.4 or -74; BSG: basigin; ?: yet-unknown receptor; Rh1, Rh2a, Rh2b, Rh4 and Rh5: reticulocyte-binding protein homologues -1, -2, 2a, -2b, -4 and -5; EBA175: erythrocyte binding antigen 175; EBA140: erythrocyte binding antigen 140; EBA181: erythrocyte binding antigen 181; EBL: erythrocyte binding ligand; Y/Z/W: unidentified receptors; CR1: complement receptor 1, GPYA: glycophorin A; GYPB: glycophorin B; GYPC: glycophorin C; Ripr: PfRh5-interacting protein, CyRPA: cysteine-rich protective antigen; AMA1: apical membrane antigen-1, DARC: Duffy antigen receptor for chemokines, RII-DBP1: Duffy binding protein 1 - region II; RBP1a/b: reticulocyte-binding protein 1a/b; RBP2a: reticulocyte-binding protein 2a; RBP2c: reticulocyte-binding protein 2c; CD71: transferrin receptor 1; RON2: rhoptry neck protein 2; RON4: rhoptry neck protein 4; RON5: rhoptry neck protein 5; AMA1 DI-II: apical membrane antigen-1 domain I-II; RON2 RI: rhoptry neck protein 2 - region I; RON2 RII: rhoptry neck protein 2 - region II.

Figure 2

DBP1 representation. (A) Schematic representation of DBP1 primary structure showing each region’s length and representing the three HARBPs located in SD3. HARBP 1639 aa sequence is shown. The highlighted residues are critical binding residues. The red dotted lines indicate the location of the Cys involved in disulfide bridges. RI (region I), RII (region 2), RIII-V (region 3 to region 5), RVI (region 6), TM (transmembrane domain), CD (cytoplasmic domain) [42,43]. (B) Alignment of RII-DBP1 from the main P. vivax strains having different geographical locations. RII-DBP1 subdomains: S1 ²¹¹N-L²⁵³ (pink), S2 ²⁷¹Y-E³⁸⁶ (gray), ³⁸⁷S3-P-S⁵⁰⁸ (cyan). DBP1 residues involved in binding to DARC: RII-DBP1 ²⁶¹F – T²⁶⁶, ²⁷⁰L – K²⁸⁹, and ³⁵⁶Q – K³⁶⁷ (The primary DARC binding interface) and ²⁵⁴V-F²⁶⁷ (secondary DARC binding interface) (black box) highlighting the critical contact residues [29,44]. Amino acid composition variations are highlighted in red. mAb 053054: ²⁶⁴D-A²⁸¹, ³⁵⁶Q-N³⁷² (red box) and 092,096 epitopes: ²⁴⁹E, ²⁷⁰L-K²⁸⁹ and ³⁵⁶Q-W³⁷⁵ (yellow box) overlapping in SD2 [28]. mAb 2D10 and 2H2 epitopes (blue box), highlighting critical contact residues (2D10-blue and 2H2-dark purple) [50]. mAb 2C6 epitope: ²⁶⁵K – F⁴⁸⁶ (dark green box), highlighting critical contact residues. mAb DB9 epitope: ⁴⁷⁶D – E⁵⁰³ (magenta box), highlighting critical contact residues [33]. mAb 3C9 epitope: ⁴⁷⁶D – E⁴⁹³ (green box), highlighting critical contact residues [55] HARBPs 1635: ³⁹⁸R – G⁴¹⁷ (purple), 1637: ⁴³⁸S – F⁴⁵⁷ (light green) and 1639: ⁴⁷⁸L –R⁴⁹⁷ (apricot) located in neutralizing epitopes’ SD3 region [56]. HARB 1639 critical binding residues matching mAb 3C9 are highlighted (arrows). HARBPs: high activity reticulocyte binding peptides. mAbs: monoclonal antibodies. Cys: cysteine.

Figure 3

RII-DBP1 structure and its interaction with DARC (A) RII-DBP1, showing its three subdomains (SD1, SD2 and SD3) and the DARC binding region [42,43]; (B) RII-DBP1 two-ligand heterotrimer structure and a monomer from the DARC receptor; (C) RII-DBP1:DARC heterotetramer structure reconstructed from PDB 3RRC, 4NUU, 4NUV, 5F3J, 6OAN, 6OAO and 6R2S [28,29,33,44,57];.(D) RII-DBP1 contact points with mAb DB9-2D10, zooming in on interacting residues highlighting HARBPs: 1635 (pink), 1637 (yellow), and 1639 (red) (PDB 6R2S and 5F3J) [33,57]; (E) DBP1/DARC points of contact with mAb 053054, zooming in on interacting residues (PBD 6OAN) [28]; (F) DBP1-DARC contact points with mAb 092096, zooming in on interacting residues (PDB6OAO) [28]; (G) RII-DBP1 including HARBPs 1625 (orange), 1627 (green), 1631 (blue), 1635 (pink), 1637 (yellow) and 1639 (red) [56].

Figure 4

Schematic representation of Plasmodium falciparum proteins. (A) Scheme for PfRh5, highlighting High Activity Binding Peptides (HABPs) location and the sequences containing residues involved in binding to BSG (top bar/box), the aforementioned residues having red arrows means PfRh5 contact sites with the basigin amino terminal extreme. PfRh5 residues contacting the BSG C-terminal are shown by brown lines and the green line show the residue contacting BSG ¹⁰²H. Green dotted lines indicate sites having polymorphism in PfRh5; red dotted lines indicate Cys location. The bottom bar/box shows PfRh5 residues contacting mAbs 9AD4 and QA5. The red arrow indicates the beginning of the sequence for HABP 36727 and 36735 participating in such contact. Residues shown in green are mAbs critical interaction residues. The complete HABP 36727 sequence is shown; residues shown in yellow underlined in black mean polymorphic sites. (B) Scheme for EBA-175, highlighting HABP location and the location of each EBA-175 region. The scheme also shows EBA-175 critical residues involved in EBA-175 dimerization (purple), those involved in interaction with glycans 1 and 2 (green), residues contacting glycans 3 and 4 (blue) and residues contacting glycans 5 and 6 (orange). Residues shown in grey are those involved in molecular interactions during complex formation. HABP 1783 (⁴³⁶H-K⁴⁵⁵) sequence shows the critical residues participating in each of the aforementioned interactions. TM (transmembrane domain), CD (cytoplasmic domain). Red shows signal peptide location in EBA-175 and PfRh5.

Figure 5

PfRh5 structure and its interactions (A) Structure of the interaction between PfRh5-BSG (PDB 4U0Q) [74], highlighting HABP binding to RBC: 36727 (green), 36728 (yellow), 36735 (orange), 36736 (blue), and 36740 (brown). HABP 36727 structure (green) determined by nuclear magnetic resonance (NMR), zooming in on residues in the interaction interface; pink shows HABP 36739 residues ⁴⁴⁸R, ⁴⁴⁹T, and ⁴⁴⁷W (close to HABP 36740), green HABP 36727 ²⁰⁷D and yellow HABP 36735 ³⁶²E; (B) Crystallographic structure of the Ripr, CyRPA and PfRh5 ternary complex in contact with the BSG receptor (PDB 6MPV) [76], zooming in on the residues belonging to the interaction interface between PfRh5-CyRPA, HABP 36736 aa ³⁹³L, and ³⁹⁷L shown in blue; (C) Rh5 crystallographic structure, showing points of contact with mAbs QA1 (PDB 4U1G) [74] and 9AD4 (PDB 4U0R) [74] highlighting HABP 36739 residues ⁴⁵¹Q and ⁴⁵²K in pink and HABP residues 36727 ²⁰⁵A, ²⁰⁹F, and ²¹²K in green.

Figure 6

EBA-175 structure and its critical interactions. (A) Crystallographic structure of EBA-175 (PDB 1ZRL) [95] highlighting HABPs 1779 (green) and 1783 (pink); (B) glycan 1 and 2 binding sites to EBA-175, HABP 1783 ⁴³⁹K and ⁴⁴²D residues contacting these glycans are highlighted in pink (PDB 1ZRO) [95]; (C) glycan 3 and 4 EBA-175 binding sites (PDB 1ZRO) [95] (D) glycan 5 and 6 EBA-175 binding (PDB 1ZRO) [95].