Figure 1.
The gp41CHRTM antigen used for immunization.
A) Schematic representation of gp41 and of the regions present in gp41CHRTM. FP, fusion peptide; HR1, N-terminal heptad repeat; C-C loop, cysteine loop, HR2, C-terminal heptad repeat; MPER, membrane proximal external region; TM, transmembrane region. The residue numbers at the domain/region boundaries are given. B) Gel filtration chromatogram of recombinant gp41CHRTM, which elutes at 13.3 ml from the column, similar to the elution profile of a marker protein of 158 kDa. This indicates that gp41CHRTM is most likely trimeric and may have an elongated structure. The inset shows a Coomassie stained SDS-PAGE gel with the gp41CHRTM protein band at the left and a protein marker at the right with the marker protein sizes in kDa indicated at the right.
Figure 2.
Amino acid and DNA sequence of 2H10.
A) Amino acid sequence. Secondary structure assignment is based on the crystal structure. β-strands are depicted as blue arrows. CDR regions are indicated with CDR1 to CDR3 and are underlined. Residues depicted in bold were mutated. Mutants that still showed binding are shown in green and mutants that do not bind to the antigen anymore are shown in red. B) DNA sequence alignment of 2H10 with the germline V-gene from which it originated. The asterisks indicate identical nucleotides. The CDR1 and CDR2 coding regions are underlined. Codons with at least two point mutations are in boldface.
Figure 3.
Epitope mapping of 2H10 and binding to helical peptides.
A) The peptide Sym-903 was used for the replacement analysis with 2F5 and 2H10. Underlined cyan colored residues are recognized by the antibodies. B) Full substitution analysis of 2H10 epitope on gp41 MPER. The binding activity of VHH 2H10 at 2 ng/ml with a peptide is shown as a vertical line proportional to the Pepscan ELISA signal. Each group of 19 lines corresponds to the replacement set of all residues except cysteine for each amino acid position in the original 15-mer cyclic peptide: KNEQELLELDKWASL. Within each group of 19 lines the substitutions are in alphabetical order: based on the one-letter amino acid code (ADEFGHIKLMNPQRSTVWY) and the reactivity of the original nonapeptide is shown as a red line. The peptides contained an N- and C-terminal cysteine and were cyclised by addition of a T2-CLIPS [115]. These results show that EQ at position 3 and 4 and LE at position 7 and 8 and K at position 11, cannot be replaced by another amino acid without loss of binding and are thus essential for binding 2H10. C) Binding of VHH 2H10 (100 ng/ml) and D) Mab 2F5 (1 ng/ml) to gp41-MPER peptides grafted in GCN4-like helical templates that contain a stabilizing Ile/Leu heptad repeat (top panel) and binding to peptides in which the zipper motif is mutated to destabilizing glycine residues (lower panel). Peptide sequences are all based on hybrids of HIV-1 gp41 and GCN4. Alignment is shown of peptide sequence with heptad repeat positions (top line). Residues essential for 2H10 binding according to the substitution analysis are underlined and residues essential for 2F5 binding are in green. Mutations are shown with one-letter codes and dots indicated that the residue is not changed.
Figure 4.
2H10, bi-2H10 and bi-2H10 W100A do not interact with membrane anchored Env.
FACS analysis of HEK293T cells transfected with JR-FL Env in its cleaved (A) and un-cleaved form (B). The increase in mean fluorescence intensity (MFI) has been blotted as a function of antibody concentration. Control antibodies include b12, b6, b12-Bio (biotinylated version of B12) and 4E10. The lack of significant b6 interaction with the cells expressing cleaved Env indicates that most Env has been processed.
Table 1.
2H10, bivalent 2H10 and mutant bi-2H10 IC50 titers against HIV-1 in TZM-bl cells.
Figure 5.
Crystal structure of 2H10 and NMR analysis of 2H10 interaction with the gp41 peptide.
A) Ribbon representation of 2H10. CDR 1, 2 and 3 are colored blue, yellow and green, respectively. Residues implicated in gp41 interaction by mutagenesis are shown as sticks. W100 at the tip of CDR3 is required for neutralization. B) Selected region of the 15N HSQC spectra (see Fig. S3) recorded on a 13C,15N labeled 2H10 sample. Increasing concentrations of the gp41 peptide were titrated into the 2H10 solution and induced specific chemical shifts; spectra were recorded with a protein∶peptide ratio of 1∶0, in grey, 1∶0.5 in blue and 1∶1 in red.C) Chemical shift perturbations (CSPs) were mapped onto the 2H10 structure. Residues showing 15N,1H chemical shift perturbations greater than 0.15 ppm are shown in red and residues with an amide resonance disappearing in the free or the bound form are colored in green.
Figure 6.
Surface plasmon resonance affinity measurements reveal essential and non-essential 2H10 residues for gp41 interaction.
A) 2H10 single domain mutants. The curves are labeled with the mutant codes in colors corresponding with the colors of the curves. The mutants R56A, R96A, E98A and R100A do not bind at all to gp140-92UG037. Fitting the curves with the two-state reaction algorithm yielded KDs of 8.0 nM for 2H10 WT and 10.1 nM for 2H10-W100A. The other fits, except for those of the non-binding mutants, yielded KDs between 5.5 nM for 2H10-S29F and 18.7 nM for 2H10-R71S B) bi-2H10-W100A mutant and wild type bi-2H10 on immobilized gp140-92UG037. Because the maximum responses units are slightly different for the two bi-2H10 molecules, the curves were normalized to the maximum response, to be able to compare the dissociation rates well.
Table 2.
Data collection and refinement statistics.
Figure 7.
Molecular modeling of the 2H10-gp41 peptide interaction suggests that 2H10 W100 is oriented towards the membrane.
A) Molecular model of the gp41 peptide interaction with 2H10 produced by HADDOCK. Salt bridges and hydrogen bonds present in the top model and found in most of the top10 models are shown as dashed lines. B) Superpositioning of the Cα atoms of the gp41 peptide derived from the HADDOCK 2H10-peptide model onto the structure of a late fusion intermediate of gp41 [29] shows that the CDR3 W100 is oriented towards the membrane and found in the same plane as gp41 residues W678, W680 and Y681. Note that there are no clashes between the 2H10 VHH and gp41.