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Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for carbohydrate binding

2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês

10.1074/jbc.ra120.014676

ISSN

1083-351X

Autores

Maria C. Bewley, L. Gautam, Mashanipalya G. Jagadeeshaprasad, D. Channe Gowda, John M. Flanagan,

Tópico(s)

Complement system in diseases

Resumo

VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria. VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria. 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VAR2CSA is a ∼350-kDa membrane protein consisting of a large ∼310-kDa nonglycosylated extracellular ectodomain, a single pass transmembrane helix, and a ∼42-kDa cytoplasmic acidic terminal sequence that interacts with a number of host cell proteins (34Salanti A. Staalsoe T. Lavstsen T. Jensen A.T. Sowa M.P. Arnot D.E. Hviid L. Theander T.G. Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria.Mol. Microbiol. 2003; 49 (12823820): 179-19110.1046/j.1365-2958.2003.03570.xCrossref PubMed Scopus (521) Google Scholar). The functional CSA-binding ectodomain is comprised of six Duffy binding-like (DBL) domains (DBL1x, DBL2x, DBL3x, DBL4ε, DBL5ε, and DBL6ε) and two interdomains (ID1 and ID2/CIDRPAM), connected to each other by short linker sequences (Fig. 1A). Together, these domains form a high affinity ligand-binding site with specificity for a minimum 12-mer CSA that has a characteristic, low C4 sulfate content (lsCSA) (31Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycans of the intervillous spaces that mediate the adherence of Plasmodium falciparum-infected erythrocytes to the placenta.J. Biol. Chem. 2000; 275 (11005814): 40344-4035610.1074/jbc.M006398200Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 33Achur R.N. Valiyaveettil M. Gowda D.C. The low sulfated chondroitin sulfate proteoglycans of human placenta have sulfate group-clustered domains that can efficiently bind Plasmodium falciparum-infected erythrocytes.J. Biol. Chem. 2003; 278 (12517756): 11705-1171310.1074/jbc.M211015200Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 37Agbor-Enoh S.T. Achur R.N. Valiyaveettil M. Leke R. Taylor D.W. Gowda D.C. Chondroitin sulfate proteoglycan expression and binding of Plasmodium falciparum-infected erythrocytes in the human placenta during pregnancy.Infect. Immun. 2003; 71 (12704116): 2455-246110.1128/iai.71.5.2455-2461.2003Crossref PubMed Scopus (27) Google Scholar). To gain structural information, small angle X-ray scattering (SAXS) studies were performed using human embryonic kidney (HEK) cell-expressed, engineered, non-N-glycosylated and baculovirus-expressed glycosylated ectodomains (38Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. et al.Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20194779): 4884-488910.1073/pnas.1000951107Crossref PubMed Scopus (111) Google Scholar, 39Clausen T.M. Christoffersen S. Dahlbäck M. Langkilde A.E. Jensen K.E. Resende M. Agerbæk M.Ø. Andersen D. Berisha B. Ditlev S.B. Pinto V.V. Nielsen M.A. Theander T.G. Larsen S. Salanti A. Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria.J. Biol. Chem. 2012; 287 (22570492): 23332-2334510.1074/jbc.M112.348839Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, the resultant molecular envelopes were relatively featureless, preventing identification of individual domains or insight into carbohydrate binding. To date, there is no high-resolution structure of the VAR2CSA ectodomain, although crystal structures of Escherichia coli-expressed DBL3x (PDB codes 3BQI and 3CML) (40Singh K. Gittis A.G. Nguyen P. Gowda D.C. Miller L.H. Garboczi D.N. Structure of the DBL3x domain of pregnancy-associated malaria protein VAR2CSA complexed with chondroitin sulfate A.Nat. Struct. Mol. 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Biol. 2013; 425 (23429057): 1697-171110.1016/j.jmb.2013.02.014Crossref PubMed Scopus (15) Google Scholar), and the tandem DBL3x-DBL4ε domains (4P1T) (44Gangnard S. Lewit-Bentley A. Dechavanne S. Srivastava A. Amirat F. Bentley G.A. Gamain B. Structure of the DBL3X-DBL4ε region of the VAR2CSA placental malaria vaccine candidate: insight into DBL domain interactions.Sci. Rep. 2015; 5 (26450557)1486810.1038/srep14868Crossref PubMed Scopus (17) Google Scholar) reveal a conserved fold comprised of three subdomains that are stabilized by multiple intradomain disulfide bonds. However, the detailed tertiary structure that forms the functional, high affinity CSA-binding surface remains unclear. It is generally agreed that domains DBL1x, ID1, DBL2x, and the N-terminal half of ID2 (ID2a) are involved (38Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. et al.Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20194779): 4884-488910.1073/pnas.1000951107Crossref PubMed Scopus (111) Google Scholar, 39Clausen T.M. Christoffersen S. Dahlbäck M. Langkilde A.E. Jensen K.E. Resende M. Agerbæk M.Ø. Andersen D. Berisha B. Ditlev S.B. Pinto V.V. Nielsen M.A. Theander T.G. Larsen S. Salanti A. Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria.J. Biol. Chem. 2012; 287 (22570492): 23332-2334510.1074/jbc.M112.348839Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 45Srivastava A. Gangnard S. Dechavanne S. Amirat F. Lewit Bentley A. Bentley G.A. Gamain B. Var2CSA minimal CSA binding region is located within the N-terminal region.PLoS ONE. 2011; 6 (21625526)e2027010.1371/journal.pone.0020270Crossref PubMed Scopus (47) Google Scholar, 46Babakhanyan A. Leke R.G. Salanti A. Bobbili N. Gwanmesia P. Leke R.J. Quakyi I.A. Chen J.J. Taylor D.W. The antibody response of pregnant Cameroonian women to VAR2CSA ID1-ID2a, a small recombinant protein containing the CSA-binding site.PLoS ONE. 2014; 9 (24505415)e8817310.1371/journal.pone.0088173Crossref PubMed Scopus (23) Google Scholar, 47Dählback M. Jørgensen L.M. Nielsen M.A. Clausen T.M. Ditlev S.B. Resende M. Pinto V.V. Arnot D.E. Theander T.G. Salanti A. The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains.J. Biol. Chem. 2011; 286 (21398524): 15908-1591710.1074/jbc.M110.191510Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); but it is unclear whether they comprise the entire surface area for CSA binding. Therefore, to understand the details of carbohydrate binding it is essential to know the structural architecture of VAR2CSA. In the present study, we have expressed and characterized, structurally and functionally, the VAR2CSA ectodomain and a set of N- and C-terminal deletion constructs (Fig. 1). These mammalian-expressed, recombinant proteins are folded and thermally stable. NTS-DBL6ε and DBL1x-ID2a specifically bind the lsCSA chains of CSPG with nanomolar affinity and ID2b-DBL6ε, DBL3x-DBL6ε, and DBL4ε-DBL6ε do not show measurable binding. Using a combination of size-exclusion chromatography in-line with SAXS (SEC-SAXS), single particle reconstruction of negative-stained electron micrographs and basic homology modeling, we have determined the relative locations of the DBL domains and produced a validated model of the VAR2CSA ectodomain. Importantly, these studies reveal, for the first time, a defined molecular shape with distinctive pores that transverse the molecule and suggest a credible model for CSA binding. The codon-optimized synthetic gene encoding the VAR2CSA ectodomain (NTS-DBL6ε) of Pf 3D7 strain and four deletion constructs corresponding to DBL1x-ID2a, ID2b-DBL6ε, DBL3x-DBL6ε, and DBL4ε-DBL6ε (as defined in Fig. 1A and Tables S1 and S2) were expressed in HEK Freestyle 293-F (HEK 293F) cells. The proteins are produced as secreted monomers and purified using a combination of ultrafiltration, nickel affinity, cation exchange, and size-exclusion chromatography. After purification and SDS-PAGE under reducing conditions, a single band at the expected apparent molecular mass was observed for each protein (Fig. 1B). Similarly, under nonreducing conditions a single band was observed for each protein (Fig. 1C). The purity of each protein construct, as assessed by the SDS-PAGE band profiles, is ∼98%. Yields of the purified recombinant proteins range from 0.5 to 0.7 mg/liter for NTS-DBL6ε to 14 mg/liter for DBL4ε-DBL6ε. Several additional deletion constructs (DBL1x-DBL5ε, DBL1-ID2b, ID1-DBL6ε, and ID1-ID2) were tested for protein production in our system, but these either failed to express or to purify as folded monomers and thus were not analyzed further. To assess the folding of the purified recombinant proteins, far UV CD spectra were recorded at 25 °C and evaluated. A strong maximum at 200 nm and troughs at 208 and 222 nm were observed (Fig. S1A) that were characteristic of proteins containing substantial α-helical secondary structures. The spectra are similar to results reported for VAR2CSA constructs DBL6ε, DBL4ε-DBL6ε, and DBL1x-DBL6ε (38Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. et al.Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20194779): 4884-488910.1073/pnas.1000951107Crossref PubMed Scopus (111) Google Scholar). The stability of each construct was investigated by following the changes in ellipticity at 222 nm as a function of temperature between 25 and 85 °C. In all cases, a transition occurs between 70 and 75 °C (Fig. S1B), demonstrating that these constructs are thermally stable. The observed transition temperatures are similar to those reported earlier for analogous constructs (38Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. et al.Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20194779): 4884-488910.1073/pnas.1000951107Crossref PubMed Scopus (111) Google Scholar). However, for each construct, the spectrum collected at 90 °C shows residual ellipticity at 208 and 222 nm (Fig. S1, C–G), demonstrating that unfolding is incomplete and preventing calculation of a thermodynamically-justified value for Tm. Indeed, the complete unfolding of NTS-DBL6ε is achieved only in the presence of the reducing agent tris(2-carboxyethyl) phosphine (Fig. S1H). These results are consistent with the constructs being stable, as expected for domains containing a large number of disulfide bonds (48Anfinsen C.B. Principles that govern the folding of protein chains.Science. 1973; 181 (4124164): 223-23010.1126/science.181.4096.223Crossref PubMed Scopus (4799) Google Scholar). To gain insight into the structural organization of the ectodomain, NTS-DBL6ε and the deletion constructs, DBL1x-ID2a, DBL3x-DBL6ε, and DBL4ε-DBL6ε, were characterized using SEC-SAXS (Table S3). The data were processed using the ATSAS Suite 3.0 (49Franke D. Petoukhov M.V. Konarev P.V. Panjkovich A. Tuukkanen A. Mertens H.D.T. Kikhney A.G. Hajizadeh N.R. Franklin J.M. Jeffries C.M. Svergun D.I. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions.J. Appl. Crystallogr. 2017; 50 (28808438): 1212-122510.1107/S1600576717007786Crossref PubMed Scopus (637) Google Scholar, 50Panjkovich A. Svergun D.I. CHROMIXS: automatic and interactive analysis of chromatography-coupled small-angle X-ray scattering data.Bioinformatics. 2018; 34 (29300836): 1944-194610.1093/bioinformatics/btx846Crossref PubMed Scopus (69) Google Scholar) and the values were obtained from this analysis compared with any published results (Table 1, Fig. 2, A–D, and Fig. S3). All of the constructs are linear in the Guinier region of the SAXS curve over the given qRg ranges (Fig. 2E), indicating that they are monodisperse. The Rg value obtained for NTS-DBL6ε is smaller than those reported previously for DBL1x-DBL6ε (38Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. et al.Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20194779): 4884-488910.1073/pnas.1000951107Crossref PubMed Scopus (111) Google Scholar, 39Clausen T.M. Christoffersen S. Dahlbäck M. Langkilde A.E. Jensen K.E. Resende M. Agerbæk M.Ø. Andersen D. Berisha B. Ditlev S.B. Pinto V.V. Nielsen M.A. Theander T.G. Larsen S. Salanti A. Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria.J. Biol. Chem. 2012; 287 (22570492): 23332-2334510.1074/jbc.M112.348839Abstrac

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