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Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

  1. Ian A. Wilsona,f,1
  1. aDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
  2. bPATH’s Malaria Vaccine Initiative, PATH Center for Vaccine Innovation and Access, Washington, DC 20001;
  3. cAtreca Inc., Redwood City, CA 94063;
  4. dDepartment of Microbiology and Immunology, Stanford University, Stanford, CA 94305;
  5. eMalaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205;
  6. fThe Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037
  1. Contributed by Ian A. Wilson, October 21, 2017 (sent for review September 7, 2017; reviewed by Brendan S. Crabb and Wim Hol)

  1. Fig. 2.

    Epitope mapping using truncation peptide arrays. (A and B) The PepSpot membrane is shown for Fab311 (A) and Fab317 (B) and consists of five rows of the spotted peptides (a1, a2, b1, b2, and c). Dark spots indicate strong Fab binding. (C and D) Schematic of the location of the peptide spots on the membrane. The numbers within the circles refer to the numbers in the truncation array sequence (D). Rows a1 and a2 correspond to a truncation array starting from the C terminus of the (NANP)6 peptide, rows b1 and b2 are truncations from the N terminus, and row c represents truncations from both the N terminus and C terminus simultaneously. The peptides that appear to have the minimal number of repeats for strong Fab binding are circled in red in A and B.

  2. Fig. 3.

    Crystal structures of (NPNA)3 peptides in complex with Fab311 and Fab317. (A and B) Surface representation of the variable domains of Fab311 (A) and Fab317 (B) with the (NPNA)3 peptide represented by a red tube. The heavy- and light-chain variable domains are colored dark and light gray, respectively. (C and D) Paratope representation for Fab311 (C) and Fab317 (D) with a transparent dark gray surface for the heavy chain and a transparent light gray surface for the light chain. The underlying CDR loops are shown in cartoon representation and are colored green (H1), blue (H2), red (H3), light green (L1), light blue (L2), and pink (L3). The (NPNA)3 peptide is shown in stick representation (yellow carbons). The N terminus of each peptide is indicated (Nterm).

  3. Fig. 4.

    Structural analysis of antibody-bound peptides. (A and D) 2Fo-Fc electron density maps contoured at 2.0σ (blue) and 0.8σ (cyan) for peptide bound to Fab311 (A) and Fab317 (D). The peptide is shown in stick representation (yellow carbons). (B and E) Type I β-turns are highlighted by transparent green circles for peptide bound to Fab311 (B) and Fab317 (E). Intrapeptide hydrogen bonds that emulate a pseudo 310 turn between the first Asn sidechain and amide backbone of the third residue in the turn are shown as black dashed lines. (C and F) Ramachandran plots for the dihedral angles of Fab311-bound peptide (C) and Fab317-bound peptide (F). Residues that have typical dihedral angles indicative of canonical NPNA type I β-turns are colored green; otherwise they are colored red. The β-sheet region is in the dark shaded region of the plot in the upper left quadrant, and the α-helical region is in the central region on the left around ψ of ?30°. The Fab311-bound and Fab317-bound peptides have one and three canonical type I β-turns, respectively.

  4. Fig. 5.

    nsEM for rsCSP bound to Fab311 and Fab317. (A and D) Five selected representative class averages for the rsCSP–Fab311 (A) and rsCSP–Fab317 (D) complexes, false colored to show the location of rsCSP (red), Fab311 (purple), and Fab317 (green). Fabs are labeled by number in white. (B) The refined 3D model confirmed the presence of multiple densities for Fab311, for which a total count of nine Fab311s could be observed at the low threshold level. The distance between the heavy-chain C termini of Fab311 nos. 2 and 3 was measured at 90 ?, which could be accommodated in an IgG. (C) The (NPNA)3 peptide (red) from the Fab311 crystal structure was docked into the 3D model (Left) and revealed a helical shape looking into the central hole of the rsCSP complex and along its length (Right). The docked peptides in the nsEM map for the rsCSP–Fab311 complex were fitted to a cylinder with a radius of 15.2 ?. The peptides are colored using a color progression, and the helical organization is shown by the dashed spiral line. (D and E) Reaching convergence for the rsCSP–Fab317 complex (E) was difficult due to the various stoichiometries seen in the 2D class averages (D). The unmodeled blob may be a remnant of a fourth Fab fragment that is present in some of the complexes.

  5. Fig. 6.

    Comparison of dihedral angles shows similarities between the bound and free peptides. (A) X-ray structure of the free ANPNA peptide shows a type I β-turn in which the Asn2 (residue i) OD1 also hydrogen bonds to the backbone amide of Asn4 (i + 2) (27). (B) Plot of the dihedral angles for the NPNA unit in the ANPNA X-ray structure; ? and ψ are shown in black and red, respectively. (C and D) Plots of the dihedral angle differences between each of the NPNA units for the peptide bound to Fab311 (C) or Fab317 (D) and the NPNA unit of the free peptide; Δ? and Δψ are shown in black and red, respectively. Type I β-turns are highlighted by transparent green boxes. The dihedral angle differences are relatively small within each NPNA type I β-turn, except for Ala9 Δψ in the Fab317 peptide (asterisk). This deviation from the NPNA type I β-turn in solution reflects a change in direction at the end of the NPNA repeat rather than a disruption of the canonical type I β-turn.

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