Skip to main content
. 2020 Nov 30;9:e59018. doi: 10.7554/eLife.59018

Figure 5. Homotypic interactions between 3D11 Fabs stabilize the 3D11 Fab-PbCSP complex.

(A and B) Close-up views of adjacent 3D11 Fabs from the cryoEM structure in complex with PbCSP (black). 3D11 Fabs bound to PbCSP form homotypic contacts with each adjacent Fab through two interfaces; one consisting of CDRs from the heavy and light chains of Fabs A and B (interface 1, A), and the second mediated by residues in FR3 of Fab A HC and FR3 of Fab C LC (interface 2, B). Variable domains of Fabs are shown in white. HCDR1, −2,–3, and KCDR1, −2 and −3 are colored yellow, orange, red, green, blue and purple, respectively. Residues forming Fab-Fab contacts are labeled with the position of the Fab in the cryoEM model (A, B or C) indicated in subscript. mAb 3D11 affinity-matured residues that engage in Fab-Fab contacts, but do not directly interact with PbCSP are highlighted in yellow with red font. Black dashed lines denote H-bonds. (C) Sequence alignment of mAb 3D11 with its inferred germline precursor. INT1 and INT2 refer to the two interfaces shown in (A) and (B). Green highlight: germline-encoded residues involved in homotypic interactions; Red: affinity-matured residues involved in homotypic interactions; Yellow highlight: affinity-matured residues involved in homotypic interactions that do not directly interact with PbCSP. (D) Binding affinity of WT 3D11 and H-58/73 germline-reverted mutant (Mut) Fabs to NPNDx1 (gray bars) and NPNDx2 (white bars) peptides as measured by ITC. Symbols represent independent measurements. Mean KD values resulting from at least two independent experiments are shown. Error bars represent standard error of the mean. An unpaired one-tailed t-test was performed using GraphPad Prism 8 to evaluate statistical significance: *p<0.05.

Figure 5.

Figure 5—figure supplement 1. Homotypic contacts between 3D11 Fabs in the 3D11 Fab-PbCSP cryoEM structure.

Figure 5—figure supplement 1.

(A) Main interaction interfaces between adjacent 3D11 Fabs in the 3D11 Fab-PbCSP cryoEM structure. (B) Table of contacts between 3D11 Fabs. HB: hydrogen bond (3.8 Å cut-off).
Figure 5—figure supplement 2. Negative-stain EM analysis of 3D11 IgG-PbCSP complexes.

Figure 5—figure supplement 2.

(A) SEC chromatogram of 3D11 IgG-PbCSP complexes. (B) Representative negative-stain (NS) micrographs of particles present in selected SEC fractions: 1 – soluble aggregates, 2 - 3D11 IgG-PbCSP complexes, 3 – unbound 3D11 IgGs. Scale bars: 100 nm. (C) Comparison of NS 2D class averages of the 3D11 IgG-PbCSP complex (upper panels) to 2D class averages of the 3D11 Fab-PbCSP complex (lower panels).
Figure 5—figure supplement 3. Comparison between cryoEM structures of 3D11 Fab-PbCSP and 311 Fab-PfCSP (PDB ID: 6MB3) (Oyen et al., 2018).

Figure 5—figure supplement 3.

CryoEM maps of the 3D11 Fab-PbCSP (A and E) and 311 Fab-PfCSP (C and G) complexes are shown as a transparent light gray surface with the CSP density highlighted in black for PbCSP and in blue for PfCSP. The CSP models built into the cryoEM maps are shown as gray for PbCSP; (B and F) or blue for PfCSP; (D) and H) sticks and aligned to the schematic representations of their respective protein sequences.