Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding

Abstract

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.

Fig. 1. Apo-structure of the ectodomain of VAR2CSA.

a SDS-PAGE non-reduced/reduced of full-length VAR2CSA (307 kDa). A gel was run after purification of VAR2CSA before the protein was aliquoted and stored at −80 °C until used. b Gel filtration profile of VAR2CSA in different buffers, top panel KCl, bottom panel NaCl. c QCM biosensor interaction between CSPG decorin and VAR2CSA, top panel KCl (k_D 0.11 nM), bottom panel NaCl (k_D1 1.4 nM). Black curves represent recorded data, red curves fitted data. d Overall structure of VAR2CSA, flipped 180° to the right. The separate domains are colored as shown in the overview below. The core region of the structure and minimal CS binding region (AA450-1025) are indicated in the legend. e α-helix from ID3 (orange) AA1955-1985 interaction with C-terminal part of ID2 (blue). To the right the structure is flipped 90° and side chains from interacting residues are shown as sticks and labeled. For clarity, helices are shown with 50% transparency. f QCM biosensor interaction between plCS and VAR2CSA in buffer with KCl (k_D 0.11 nM). Black curves represent recorded data, red curves fitted data. Source data are provided in source data file.

Fig. 2. Structural analysis of DBL2 and CS binding groove.

a Comparison of domains DBL2, DBL3 and DBL4. The red loop shows the “WIW-motif”-loop from each domain, the green α-helix shows HB1. In DBL2, an additional loop in HB1 introduces a kink in the α-helix. b DBL2 domain shown as cartoon, the red loop shows S1 (HB4 and “WIW-motif”-loop), blue α-helices show S2 (HB3-HB5) and green α-helices show S3 (HB2-HB1). S1-S3 residue boundaries and colors are shown in legend below. The distal nitrogen atom of Lys/Arg residues side chains in binding groove are shown as blue spheres and labeled according to residue number. c Structural details of the interaction between “WIW-motif” and HB2/HB3 in DBL2. Interacting side chains are shown as sticks and are labeled with residue numbers. d LOGO conservation analyses of “WIW-motif”-loop AA553-583 in var2csa variants. Residue number from VAR2CSA sequence shown below.

Fig. 3. Hydroxyl radical protein foot printing and CS binding analysis of DBL1-ID2 mutant.

a FPOP analysis of placental CS binding to wild-type DBL1-ID2. 13 chymotryptic peptides in VAR2CSA were found to exhibit significant protection from oxidation upon binding to placental CS (blue asterisks, p < 0.05, two-tailed Student’s t test). Two peptides (red asterisks, p < 0.05, two-tailed Student’s t test) exhibit significant exposure. Peptides not detected as oxidized in either condition are not shown. b SDS-PAGE non-reduced/reduced of DBL1-ID2 wild-type (KKKWIWKK) and mutant (KKKAIAKK). The gel shown is from one purification, the mutant DBL1-ID2 was purified three times and the wild-type protein more than three times. c T_m determination by nanoDSF melting curves of wild-type (T_m 67.7 °C) and mutant (T_m 63.7 °C) DBL1-ID2. Region between 40 °C and 80 °C is shown. d QCM biosensor kinetic measurements of wild-type (top, k_D1 1.1 nM) and mutant DBL1-ID2 (bottom, no binding) to decorin CSPG. e Flow cytometry binding analyses of lung cancer cell line A549 to wild-type and mutant DBL1-ID2. Source data are provided in source data file.

Fig. 4. Docking model of VAR2CSA in complex with a CS 20-mer.

a The docking structure of VAR2CSA in complex with CS 20-mer. The electrostatic potential surfaces of VAR2CSA with the minimal binding region and binding groove, which is highlighted by a black box. A surface potential (kT/e) color bar is shown. The CS molecule is represented by sticks in red. The DBL5/6 domains are shown in white cartoon. The right panel shows the structure turned 90°. b Docking structure of VAR2CSA shown as cartoon with domains colored as in Fig. 1d. The CS molecule is shown as sticks in magenta. c Conformational flexibility of CS ligand in the binding groove of VAR2CSA. Boxed area shows the core part of the CS chain with relatively low RMSF values, corresponding to roughly seven disaccharide units. d Binding groove of VAR2CSA with an ensemble of CS 20-mer with 70 conformers sampled in a 70 ns MD simulation with an interval of 1 ns. CS interacting residues from VAR2CSA are labeled with residue numbers.