Inhibitory antibodies identify unique sites of therapeutic vulnerability in rhinovirus and other enteroviruses

Figures

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  Fig. 1.. YDF and GGVV bind to different epitopes on HRV14 3C protease. (A) Crystal structure of HRV14 3C in complex with YDF scFv, with HRV14 3C colored in light gray and VH and VL of YDF scFv colored in cyan and blue, respectively. Both the heavy-chain and light-chain CDRs on YDF participate in interaction with HRV14 3C. (B) Structure of HRV14 3C in complex with GGVV scFv, with HRV14 3C colored in dark gray and the VH and VL of YDF scFv colored in green and lime, respectively. Only the heavy chain of GGVV is involved in binding HRV14 3C. (C) Superimposition of the two complexes, color-coded as above. (D) YDF and GGVV bind to different surfaces on HRV14 3C, both of which are distal to the active site. The contact regions of YDF VH and VL on HRV14 3C are highlighted in cyan and blue, respectively, and the epitope of GGVV on HRV14 3C is highlighted in green. The three critical residues in the active site of HRV14 3C are shown as a red surface.

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  Fig. 2.. YDF allosterically inhibits the endopeptidase activity of HRV14 3C. (A) The protein–protein interface between HRV14 3C and YDF. HRV14 3C is shown as gray ribbons, with loop 107 to 114 and loop 138 to 145 highlighted in purple and pink, respectively. CDRH3 and the three light-chain CDRs of YDF that are involved in binding HRV14 3C are highlighted in cyan and blue, respectively. (B) Close-up views of the interface between CDRH3 (YDF) and HRV14 3C (Top) and the interface between CDRLs (YDF) and HRV14 3C (Bottom). Key residues that are involved in hydrophilic or hydrophobic interactions on protein–protein interfaces are shown as stick models and color-coded as in A. Polar and electrostatic interactions are indicated with black dashed lines. (Bottom) Electrostatic surface of HRV14 3C is shown to illustrate the hydrophobic cage that is formed by Y139, A140, K142, and F108 on HRV14 3C. Hydrophobic surfaces are in whitish gray, basic in blue, and acidic in red. The CDRs residues are labeled following Kabat numbering. (C) Protease activity (micromoles per second) of HRV14 3C was plotted against the concentration of substrate (micromolar) in the absence or presence of different molar…

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  Fig. 3.. GGVV binds HRV14 3C only with two heavy-chain CDRs. (A) The protein–protein interfaces between HRV14 3C protease and GGVV. The three critical residues are shown as red stick models to illustrate the position of active site. HRV14 3C is shown as gray ribbons, while helix α1, 310 helix η2, and the β-turn between the β10 and β11 strands at the antibody–protein interface are highlighted in orange. CDRH3 and CDRH2 of GGVV that are involved in binding HRV14 3C are highlighted in green. (B and C) Close-up views of the interfaces between CDRH2 (GGVV) and HRV14 3C (B) and between CDRH3 (GGVV) and HRV14 3C (C). HRV14 3C is shown as an electrostatic surface, with helix α1, 310 helix η2, and the β-turn between the β10 and β11 strands as orange ribbons. Key residues that are involved in interactions at the antibody–protein interface are shown as stick models, labeled and color-coded as in A. Polar and electrostatic interactions are indicated with black dashed lines. The CDR residues of GGVV are labeled following Kabat numbering.

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  Fig. 4.. GGVV blocks HRV14 3C binding to HRV genomic RNA. (A) The structure of HRV14 3C is shown as a surface representation, onto which the HDX difference heat map for HRV14 3C in complex with 72-nt RNA compared to the apo form is mapped. For heat-map color coding, red indicates increased HDX and blue indicates decreased HDX in 3C-RNA relative to apo 3C, light gray indicates no significant changes, and dark gray represents regions that were not detected in the HDX experiment. CDRH2 and CDRH3 of GGVV are shown as green stick models to illustrate the paratopes of GGVV. (B–D) Deuterium uptake plots and mass spectra of indicated peptides from HRV14 3C in the absence and presence of 72-nt RNA. Note that the HDX in peptides 9 to 16, 85 to 93, and 151 to 160 decreased considerably in the presence of 72-nt RNA, suggesting that residues in these peptides are located at the interface between HRV14 3C and 72-nt RNA. (Left) The deuterium uptake data are plotted as percent deuterium uptake verse time on a logarithmic scale. (Right) Mass spectra of indicated peptides at different labeling time points, with the mass spectra of undeuterated samples shown as controls. (E) ITC results of RNA titrated into…

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  Fig. 5.. Key residues on 3C involved in both GGVV and genomic RNA interactions are conserved across a panel of enteroviruses. (A) Sequence alignment of HRV14 3C with its orthologs from HRV2, EV93, EV71, CVB3, and CVA16. The secondary structure of HRV14 3C is shown above the sequence and the three critical active-site residues are indicated with orange stars. Residues on HRV14 3C involved in both GGVV and 72-nt RNA binding are marked by red triangles, and residues involved in GGVV binding but not 72-nt RNA binding are marked by light gray triangles. (B) The structure of HRV14 3C (gray) is shown as a cartoon representation onto which its orthologs from HRV2 (PDB ID code 5FX5), EV93 (PDB ID code 3Q3X), EV71 (PDB ID code 3SJK), CVB3 (PDB ID code 2ZTZ), and CVA16 (PDB ID code 3SJ8) are superimposed. The backbone of the residues that are marked by red triangle in A are shown as stick models to highlight their structural conservation in space across HRV14 (gray), HRV2 (cyan), EV93 (orange), EV71 (green), CVB3 (blue), and CVA16 (hot pink). (C) The 3C proteases from HRV14, HRV2, EV93, EV71, CVB3, and CVA16 were rendered as an electrostatic surface representation (−5 to +5 kT/e), where hydrophobic…