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

doi:10.21203/rs.3.rs-121821/v1

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

https://doi.org/10.21203/rs.3.rs-121821/v1

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published 19 May, 2021

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Malaria during pregnancy remains a major health problem in Plasmodium falciparum endemic areas. 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. Placental malaria can have severe consequences for both mother and child by causing maternal anemia, low birth weight and stillbirth. Several VAR2CSA-based vaccines have been developed and clinically tested but they have failed to induce an antibody response that effectively inhibits placental adhesion of different genetic variants of VAR2CSA. The interaction between VAR2CSA and CS represents a unique case of an evolution-driven high-affinity interaction between a protein and an oncofetal carbohydrate. Here, we report cryo-EM structures of the VAR2CSA ectodomain 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 enable rational design of second-generation placental malaria vaccines eliciting broadly VAR2CSA-reactive antibodies and novel cancer therapies.

Parasitology

Applied & Industrial Microbiology

General Microbiology

placental malaria VAR2CS

cryo-EM

Yes there is potential Competing Interest. AS, TGT, MAN, TMC are listed as co-inventors on a patent family covering the use of VAR2CSA to target and diagnose cancer. AS and TGT are listed as co-inventor on a patent on using VAR2CSA as a prophylactic malaria vaccine during pregnancy. J.S.S. has financial interest in an early-stage company commercializing technologies for protein higher order structure analysis. The other authors have no financial conflicts of interest.

finalTable1.pdf
Table S1: Cryo-EM data collection data processing and model building statistics.
FigureS1.pdf
Fig S1: Resolution and structural flexibility of determined the cryoEM structure a) CryoEM map of apo VAR2CSA, colored by local resolution at two different level. b) Cryo-EM map of the VAR2CSA in presence of plCS, colored by local resolution at two different levels. c) The VAR2CSA-plCS map with DBL1/5/6 part partially transparent and show at a different contour level compared to the core structure. Compared to the apo structure, the core part is conformationally stable, while DBL1/5/6 are flexible. d) Two independent maps/models calculated using separate sub-fractions of particles. Alignment using the core structure shows displaced are DBL5/6.
FigureS2.pdf
Fig S2: Quality of the cryo-EM density in selected functionally important regions a) ID3-helix (residue AA1955-1985). b) C-terminal part of ID2 which is interacting with ID3 α-helix (residue AA1140-1181). c) “WIW-motif”-loop in DBL2 (residue AA556-590). d) HB1 C-terminal half of α-helix in DBL2 involved in CSA binding (residue AA758-783)
FigureS3.pdf
Fig S3: a) LOGO conservation of ID3 region (residues AA1955-1985) in VAR2CSA subvariants. b) LOGO conservation of ID2 region interacting with ID3 (residues AA1139- 1181).
FigureS4.pdf
Fig S4: The cryo-EM structure of the DBL3/4 domains compared to the equivalent crystal structure (PDB-ID 4P1T). The crystal structure is colored in gray and the cryo-EM structure of DBL3 in purple and DBL4 in yellow.
FigureS5.pdf
Fig S5: Workflow of the cryo-EM structure determination of the VAR2CSA apo structure. a) Representative micrograph. b) Representative 2D class averages with a box size of 36.6nm. c) Brief flowchart of the data processing. The Methods section and Table S1 contains further details. d) Gold standard Fourier shell correlation (FSC) curve of the map. e) FSC curve of build model vs map. f) The final map colored by resolution.
FigureS6.pdf
Fig S6: Workflow of the cryo-EM structure determination of the VAR2CSA plCS structure.a) Representative micrograph. b) Representative 2D class averages with a box size of 36.6nm.c) Brief flowchart of the data processing. The Methods section and Table S1 contains further details. d) Gold standard Fourier shell correlation (FSC) curve of the map. e) FSC curve of build model vs map. f) The final map colored by resolution.
FigureS7.pdf
Fig S7: LOGO plot of VAR2CSA ectodomain. Domain and homology block borders indicated above LOGO along with data from FPOP analysis of CS-protected/exposed peptides in the DBL1-ID2 protein and CS-interacting residues predicted by Molecular Docking (MD). Small LOGO alignment below in the middle shows atypical HBs in VAR2CSA DBL2. Graphic HB LOGO legend modified from10.
FigureS8.pdf
Fig S8: FPOP-MS peptides mapped on the surface of the VAR2CSA apo structure with docked CSA 20-mer shown as purple sticks. Cyan regions indicate an up to 2x reduction in oxidation and blue regions show more than 2x reduction. Red regions designate an increase in surface exposure when plCS is bound.
FigureS9.pdf
Fig S9: Domain flexibility revealed by MD simulations. The RMS fluctuation of VAR2CSA at a residue level revealed by the MD simulation of the VAR2CSA-CS complex. There are significant motions observed in DBL1/5/6 domains and also the loop regions as illustrated by the grey transparent tubes.

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

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