1. Structural Biology and Molecular Biophysics

Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats

  1. Iga Kucharska
  2. Lamia Hossain
  3. Danton Ivanochko
  4. Qiren Yang
  5. John L Rubinstein
  6. Régis Pomès
  7. Jean-Philippe Julien  Is a corresponding author
  1. Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Canada
  2. Department of Biochemistry, University of Toronto, Canada
  3. Department of Medical Biophysics, University of Toronto, Canada
  4. Department of Immunology, University of Toronto, Canada
https://doi.org/10.7554/eLife.72908
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Cite this article
  1. Iga Kucharska
  2. Lamia Hossain
  3. Danton Ivanochko
  4. Qiren Yang
  5. John L Rubinstein
  6. Régis Pomès
  7. Jean-Philippe Julien
(2022)
Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats
eLife 11:e72908.
https://doi.org/10.7554/eLife.72908
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7 figures, 2 tables and 3 additional files

Figures

Figure 1
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Comparison of PvCSPvk210 and PvCSPvk247 repeat sequences.

(A) Schematic representations of PvCSPvk210 and PvCSPvk247 sequences, each including an N-terminal domain, central repeat region, and C-terminal domain. Colored blocks represent repeat motifs. The …

Figure 2 with 2 supplements
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Conformational flexibility of PvCSPvk210 and PvCSPvk247 peptides.

(A) Superposition of the conformations of peptides resulting from molecular dynamics (MD) simulations at every 2 ns and aligned to the conformational median structure. (B) Example snapshots of the …

Figure 2—figure supplement 1
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Time evolution of structural properties of PvCSP peptides.

Properties are averaged over 20 replicas, with the shading area representing standard error of the mean. (A) H-bond turn propensity (Xturn). (B) Number of peptide-peptide H-bonds per residue (XHB).

Figure 2—figure supplement 2
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Ensemble-averaged backbone-backbone H-bonding maps for each PvCSP peptide sequence.

A forward H-bond forms between C=O (acceptor) of residue i and N–H (donor) of residue i + n (upper diagonal), while the reverse forms a reverse H-bond (lower diagonal). Within the diagonal …

Figure 3 with 4 supplements
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2F2 Fab binding to PvCSPvk210 repeat peptides.

(A) Affinities of 2F2 Fab for peptides 210-1, 210-2, 210-3, 210-4, and 210-5 as measured by isothermal titration calorimetry (ITC). Open circles represent independent measurements. Mean binding …

Figure 3—figure supplement 1
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Isothermal titration calorimetry (ITC) measurements of 2F2 Fab binding to peptides 210-1 (A), 210-2 (B), 210-3 (C), 210-4 (D), and 210-5 (E).
Figure 3—figure supplement 2
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Stereo-image of the composite omit map electron density contoured at 1.0–1.2 sigma for peptides 210-1 (A), 210-2 (B), 210-3 (C), 210-4 (D), and 210-5 (E).
Figure 3—figure supplement 3
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Interactions between germline-encoded aromatic residues in anti-CSP monoclonal antibodies (mAbs) and repeat peptides.

Heavy chain complementarity-determining region (HCDR) and kappa chain complementarity-determining region (KCDR) are colored green and gray, respectively. The positions of germline-encoded tryptophan …

Figure 3—figure supplement 4
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Electrostatic surface potential of monoclonal antibodies (mAbs) 2F2 (A), 2E10.E9 (B), 1210 (Imkeller et al., 2018) (C), and 3D11 (Kucharska et al., 2020) (D) bound to CSP peptides.

Scale: –5 kT e–1 (red) to +5 kT e–1 (blue).

Figure 4 with 2 supplements
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2E10.E9 Fab binding to PvCSPvk247 repeat peptides.

(A) Affinities of 2E10.E9 Fab for peptides 247-1, 247-2, 247-3, and 247-4 as measured by isothermal titration calorimetry (ITC). Open circles represent independent measurements. Mean binding …

Figure 4—figure supplement 1
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Isothermal titration calorimetry (ITC) measurements of 2E10.E9 Fab binding to peptides 247-1 (A), 247-2 (B), 24-3 (C), and 247-4 (D).
Figure 4—figure supplement 2
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Stereo-image of the composite omit map electron density contoured at 1.2 sigma for peptides 247-2 (A), 247-3 (B), and 247-4 (C).
Figure 5
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Homotypic Fab-Fab interactions in Fab 2E10.E9-247-2 peptide complex.

(A, B) 2E10.E9 Fabs that simultaneously recognize the 247-2 peptide contact each other through an interface consisting of mainly of heavy chain complementarity-determining region (HCDR)2 of both Fab …

Figure 6 with 1 supplement
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Binding of 2F2 and 2E10.E9 to full-length PvCSPvk210 and PvCSPvk247.

Binding kinetics of twofold dilutions of 2F2 IgG and Fab (A, upper panel and lower panel, respectively) to PvCSPvk210, and 2E10.E9 IgG and Fab (B, upper panel and lower panel, respectively) to …

Figure 6—source data 1

SDS-PAGE analysis of 2F2 Fab-PvCSPvk210 and 2E10.E9 Fab-PvCSPvk247 complexes.

https://cdn.elifesciences.org/articles/72908/elife-72908-fig6-data1-v2.zip
Figure 6—figure supplement 1
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Negative stain electron microscopy (NS EM) and electron cryomicroscopy (cryo-EM) analysis of 2E10.E9 Fab-PvCSPvk247 (A) and 2F2 Fab-PvCSPvk210 (B) complexes.

Upper panels show representative NS EM (left panel) and cryo-EM (right panel) micrographs. Positions of representative individual particles are highlighted with white circles. Lower panels: …

Figure 7
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Evaluation of flexibility for various Fab-CSP peptide complexes by negative stain electron microscopy.

Refined 3D classes (upper panels) and representative 2D class averages (bottom panels) of (A) 2F2 Fab-210-10 peptide, (B) 2E10.E9 Fab-247-2 peptide, (C) 3D11 Fab-NPNDx2 peptide (PPPPNPND)3 (Kucharska…

Tables

Table 1
X-ray crystallography data collection and refinement statistics.
2F2-210-12F2-210-22F2-210-32F2-210-42F2-210-52E10-247-22E10-247-32E10-247-4
BeamlineAPS 23-ID-BAPS 23-ID-DAPS 23-ID-BAPS 23-ID-BAPS 23-ID-DAPS 23-ID-BAPS 23-ID-BAPS 23-ID-D
Wavelength (Å)1.0331671.0331671.0331671.0331671.0331671.0331671.0331671.033200
Space groupP 1C 2C 2P 1C 2P 31P 21P 1
Cell dimensions71.5, 81.4, 82.392.9, 60.4, 158.392.6, 60.8, 81.471.7, 82.3, 82.893.4, 60.5, 159.1142.4, 142.4, 91.356.4, 144.4, 60.554.5, 66.3, 142.3
α, β, γ (o)94.6, 114.1, 111.690, 101.5, 9090, 101.6, 9095.3, 113.8, 111.590, 101.2, 9090, 90, 12090, 102.8, 90100.4, 92.3, 91.7
Resolution (Å)*29.48–2.20 (2.25–2.20)29.21–2.54 (2.65–2.54)29.71–1.97(2.02–1.97)29.61–2.67 (2.77–2.67)29.69–2.27 (2.34–2.27)29.55–3.19 (3.30–3.19)29.48–2.68 (2.78–2.68)29.34–2.71 (2.81–2.71)
No. molecules in the asymmetric unit (ASU)32132224
No. observations264,344 (16,542)181,285 (21,112)101,891 (4384)155,413 (16,123)471,275 (38,772)693,999 (70,530)184,740 (18,748)552,431 (55,667)
No. unique observations75,088 (4440)28,642 (3484)31,038 (1962)43,179 (4542)40,512 (3722)34,411 (3469)26,439 (2627)53,126 (5316)
Multiplicity3.5 (3.7)6.3 (6.1)3.3 (2.2)4.7 (1.5)11.6 (10.3)20.1 (20.3)7.0 (7.1)10.4 (10.5)
Rmerge (%)†15.5 (95.2)16.4 (144.5)6.5 (55.7)13.9 (62.8)34.2 (175.0)36.1 (370.6)21.6 (158.6)22.4 (123.5)
Rpim (%) ‡7.5 (32.4)10.6 (97.6)6.1 (46.9)7.8 (30.8)15.3 (83.0)8.2 (84.0)8.8 (63.7)7.3 (39.8)
< I/σ I>5.2 (1.5)6.7 (1.5)8.3 (1.5)4.7 (1.5)9.4 (1.5)13.2 (1.7)9.2 (1.6)7.4 (1.6)
CC1/20.965 (0.527)0.994 (0.529)0.996 (0.603)0.987 (0.669)0.927 (0.435)0.998 (0.756)0.993 (0.578)0.995 (0.729)
Completeness (%)97.5 (97.1)99.8 (100.0)98.7 (90.5)98.3 (97.8)99.9 (100.0)99.8 (99.6)99.9 (100.0)100.0 (100.0)
Refinement statistics
Reflections used in refinement74,67228,60431,03043,16040,50134,41126,43953,126
Reflections used for R-free37311432155221622026173213142067
Non-hydrogen atoms10,5806872359510,50469416818688513,707
Macromolecule10,1236864341810,34468416818684513,620
Water4338177160100-3487
Heteroatom------6-
R §work/Rfree17.9/22.020.2/24.918.6/23.518.6/22.819.2/23.818.0/21.020.7/23.920.9/24.6
Rms deviations from ideality
Bond lengths (Å)0.0070.0020.0150.0020.0030.0100.0100.010
Bond angle (°)0.870.511.330.570.711.391.251.22
Ramachandran plot
Favored regions (%)97.597.997.397.998.694.697.197.5
Allowed regions (%)2.32.12.71.91.45.42.92.5
B-factors (A2)
Wilson B-value39.164.735.547.050.091.042.154.7
Average B-factors45.775.045.751.053.7111.071.077.0
Average macromolecule45.775.945.951.153.7111.071.477.2
Average heteroatom------69.8-
Average water molecule44.061.441.141.954.3-37.843.3
  1. *

    Values in parentheses refer to the highest resolution bin.

  2. †

    Rmerge = ∑hkl ∑i | Ihkl, i -< Ihkl > | / ∑hkl< Ihkl > .

  3. ‡

    Rpim = ∑hkl [1/(N – 1)]1/2 ∑i | Ihkl, i -< Ihkl > | / ∑hkl< Ihkl > .

  4. §

    Rwork = (∑ | |Fo | - |Fc | |) / (∑ | |Fo |).

  5. 5% of data were used for the Rfree calculation.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Recombinant DNA reagentpcDNA3.4-2F2
Fab HC (plasmid)		This paperN/A2F2 Fab heavy chain gene in pcDNA3.4 TOPO vector
Recombinant DNA reagentpcDNA3.4-2F2
KC (plasmid)		This paperN/A2F2 light chain gene in pcDNA3.4 TOPO vector
Recombinant DNA reagentpcDNA3.4-2E10.E9
Fab HC (plasmid)		This paperN/A2E10.E9 Fab heavy chain gene in pcDNA3.4 TOPO vector
Recombinant DNA reagentpcDNA3.4-2E10.E9
KC (plasmid)		This paperN/A2E10.E9 light chain gene in pcDNA3.4 TOPO vector
Recombinant DNA reagentpcDNA3.4-
PvCSPvk210-
His6x (plasmid)		This paperN/APvCSPvk210 gene with His tag in pcDNA3.4 TOPO vector
Recombinant DNA reagentpcDNA3.4-
PvCSPvk247-
His6x (plasmid)		This paperN/APvCSPvk247 gene with His tag in pcDNA3.4 TOPO vector
Cell line (Homo sapiens)FreeStyle
293F cells		Thermo Fisher ScientificCat# R79007 
Cell line (Mus musculus)2F2
hybridoma cell line		Nardin et al., 1982, Alan Cochrane, unpublished resultsBEI Resources #MRA-184; RRID:CVCL_A7VR 
Cell line (M. musculus)2E10.E9
hybridoma cell line		Nardin et al., 1982, Alan Cochrane, unpublished resultsBEI Resources #MRA-185; RRID:CVCL_A7VT 
Chemical compound, drugGibco FreeStyle
293 Expression
Medium		Thermo Fisher ScientificCat# 12338026 
Chemical compound, drugGibco
Hybridoma-SFM		Thermo Fisher ScientificCat# 12045076 
Chemical compound, drugFectoPRO DNA
Transfection
Reagent		VWRCat# 10118-444 
Chemical compound, drugFetal bovine
serum		Thermo Fisher ScientificCat# 12483-020 
Antibody2F2 IgG
(mouse
monoclonal)		Nardin et al., 1982, Alan Cochrane, unpublished resultsN/APurified from 2F2 hybridoma cell line; see Materials and methods
Antibody2E10.E9 IgG
(mouse
monoclonal)		Nardin et al., 1982, Alan Cochrane, unpublished resultsN/APurified from 2E10.E9 hybridoma cell line; see Materials and methods
Biological sample (Carica papaya)Papain from
papaya latex		Sigma-AldrichCat# P4762 
Peptide, recombinant protein1210 FabImkeller et al., 2018N/ASee Materials and methods for concentrations and masses used, and buffer conditions
Peptide, recombinant protein3D11 FabKucharska et al., 2020N/ASee Materials and methods for concentrations and masses used, and buffer conditions
Peptide, recombinant protein2F2 FabThis paperN/ASee Materials and methods for concentrations and masses used, and buffer conditions
Peptide, recombinant protein2E10.E9 FabThis paperN/ASee Materials and methods for concentrations and masses used, and buffer conditions
Peptide, recombinant protein210-1
(GDRADGQ
PAGDRADGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-2
(GDRAAGQ
PAGDRAAGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-3
(GDRADGQP
AGDRAAGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-4
(GDRAAGQ
PAGDRADGQP)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-5
(GDRAAGQ
PAGNGAGGQAA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-6
(GDRADGQ
PAGDRADGQ
PAGDRADGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-7
(GDRAAGQ
PAGDRAAGQ
PAGDRAAGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-8
(GDRADGQ
PAGDRAAGQ
PAGDRADGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-9
(GDRAAGQ
PAGDRAAGQ
PAGNGAGGQAA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein210-10
(GDRADGQ
PAGDRADGQ
PAGDRADGQ
PAGDRADGQPA)		This paperN/ADerived from PvCSPvk210 repeat region
Peptide, recombinant protein247-1
(ANGAGNQ
PGANGAGNQ
PGANGAGNQPG)		This paperN/ADerived from PvCSPvk247 repeat region
Peptide, recombinant protein247-2
(EDGAGNQ
PGANGAGNQ
PGANGAGNQPG)		This paperN/ADerived from PvCSPvk247 repeat region
Peptide, recombinant protein247-3
(ANGAGNQ
PGANGAGNQ
PGANGAGGQAA)		This paperN/ADerived from PvCSPvk247 repeat region
Peptide, recombinant protein247-4
(ANGAGNQ
PGANGAGNQPG)		This paperN/ADerived from PvCSPvk247 repeat region
Peptide, recombinant proteinNPNDx2
(PPPPNPNDP
PPPNPNDP
PPPNPND)		Kucharska et al., 2020N/ADerived from PbCSP ANKA repeat region
Peptide, recombinant proteinNANP5
(NANPNAN
PNANPNA
NPNANP)		Imkeller et al., 2018N/ADerived from PfCSP NF54 repeat region
Software, algorithmGROMACS 2016.5Abraham et al., 2015; Berendsen et al., 1995https://manual.gromacs.org/documentation/2016-current/index.html;RRID:SCR_014565 
Software, algorithmCHARMM22*Best and Hummer, 2009; Best and Mittal, 2010; Lindorff-Larsen et al., 2012; MacKerell et al., 1998; Piana et al., 2011https://www.charmm.org/charmm/?CFID=66837e22-4ee5-47ba-bcbf-b4b385c2397e&CFTOKEN=0; RRID:SCR_014892 
Software, algorithmLINCSHess, 2008N/A 
Software, algorithmParticle-Mesh
Ewald algorithm		Darden et al., 1993; Essmann et al., 1995N/A 
Software, algorithmParrinello–Rahman algorithmParrinello and Rahman, 1981N/A 
Software, algorithmVMDHumphrey et al., 1996https://www.ks.uiuc.edu/Research/vmd/; RRID:SCR_001820 
Software, algorithmMatplotlibHunter, 2007https://matplotlib.org/; RRID:SCR_008624 
Software, algorithmMDTrajMcGibbon et al., 2015https://www.mdtraj.org/1.9.5/index.html 
Software, algorithmOctet Data AnalysisSoftware 9.0.0.6ForteBiohttps://www.fortebio.com/products/octet-systems-software 
Software, algorithmMicroCal ITC Origin7.0 Analysis SoftwareMalvernhttps://www.malvernpanalytical.com/ 
Software, algorithmASTRAWyatthttps://www.wyatt.com/products/software/astra.html; RRID:SCR_016255 
Software, algorithmGraphPad Prism 8GraphPad Softwarehttps://www.graphpad.com/; RRID:SCR_002798 
Software, algorithmSBGridSBGrid Consortiumhttps://sbgrid.org/; RRID:SCR_003511 
Software, algorithmcryoSPARC v2Punjani et al., 2017https://cryosparc.com/; RRID:SCR_016501 
Software, algorithmRelionScheres, 2012https://www3.mrc-lmb.cam.ac.uk/relion/; RRID:SCR_016274 
Software, algorithmXDSKabsch, 2010http://xds.mpimf-heidelberg.mpg.de/; RRID:SCR_015652 
Software, algorithmPhaserMcCoy et al., 2007https://www.phenix-online.org/; RRID:SCR_014224 
Software, algorithmPhenix (phenix.
refine; phenix.real_
space_refine)		Adams et al., 2010https://www.phenix-online.org/; RRID:SCR_014224 
Software, algorithmUCSF ChimeraPettersen et al., 2004https://www.cgl.ucsf.edu/chimera/; RRID:SCR_004097 
Software, algorithmUCSF ChimeraXGoddard et al., 2018https://www.cgl.ucsf.edu/chimerax/; RRID:SCR_015872 
Software, algorithmCootEmsley et al., 2010https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/; RRID:SCR_014222 
Software, algorithmPyMOLThe PyMOL Molecular Graphics System, version 1.8 Schrödinger, LLC.https://pymol.org/2/#products; RRID:SCR_000305 
Software, algorithmPDBePISAKrissinel and Henrick, 2007https://www.ebi.ac.uk/pdbe/pisa/; RRID:SCR_015749 
Software, algorithmStrideHeinig and Frishman, 2004http://webclu.bio.wzw.tum.de/stride/ 
OtherHomemade
holey gold grids		Marr et al., 2014N/A 
OtherHomemade
carbon grids		Booth et al., 2011N/A 

Additional files

Supplementary file 1

Summary of CSP-derived peptides used in this study.

https://cdn.elifesciences.org/articles/72908/elife-72908-supp1-v2.docx
Supplementary file 2

Intramolecular H-bonds (3.0 A cutoff) in PvCSP peptides observed in Fab-peptide co-crystal structures.

No intramolecular H-bonds were detected for peptide 247-4.

https://cdn.elifesciences.org/articles/72908/elife-72908-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/72908/elife-72908-transrepform1-v2.pdf

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