Journal: Nature structural & molecular biology

Article Title: Structure-based energetics of protein interfaces guide Foot-and-Mouth Disease virus vaccine design

doi: 10.1038/nsmb.3096

Figure Lengend Snippet: FMDV capsids readily fall apart into pentameric assemblies. (a) Surface representation of atomic models of the intact FMDV serotype O capsid (left panel; PDB 1BBT) and of its dissociation into twelve pentameric assemblies upon storage, heating or lowering pH (right panel). VP1, VP2, VP3 and VP4 are labelled and coloured blue, green, red and yellow, respectively. (b) Negative stain EM images of the inactivated O1M wild-type capsids soon after purification (left panel; intact with only a few detectable pentamers) or after 10 days of storage at 4°C (right panel; 80% dissociated into pentamers). The scale bar indicates 50nm. Class averages of the intact capsid or pentamers are shown in the zoom windows (each calculated from 700 particles using EMAN238). The scale bar indicates 5nm. (c) Cartoon representation of the atomic structure of the O1M model showing two icosahedral protomers forming an inter-pentameric interface (left panel; VP1, VP2, VP3 and VP4 are labelled and coloured blue, green, red and yellow, respectively). A truncated model was generated by trimming the protomers to include VP2 and VP3 atoms within 13Å of the interface as shown in (d). Residues on 2-fold symmetry-related helices for O, A and SAT serotypes are highlighted in right panel. (d) 1.5ns MD trajectory showing a large RMSD from the starting structure for an unrestrained model (average RMSD 1.83 Å2) in the left panel, with the restrained MD trajectory shown in the right panel (dummy atoms shown in green are placed at the midpoints of the inter-protomeric interface to define the restraints). The beginning of the trajectory is shown in red, the middle in white and the end in blue.

Article Snippet: The tyrosine density for O1M S93Y was also observed but it was less well defined due to the twinning of O1M crystals. ( e ) Cryo-EM maps showing the density for S93Y for O1M and SAT2 at 3.2Å and 3.5Å resolution, respectively. table ft1 table-wrap mode="anchored" t5 caption a7 O1M VP2 S93Y (a) A22 VP2 H93F (b) Data collection Space group I23 I222 Cell dimensions a , b , c (Å) 344.08, 344.08, 344.08 327.6, 341.3 363.6 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 46.0 – 3.5 (3.56 – 3.5) c 50.0 – 2.4 (2.49 - 2.4) R merge 0.46 0.27 I / σ I 1.7(0.76) 1.23 (0.5) Completeness (%) 77.7 (71.1) 45.5 (39.8) Redundancy 1.5(1.4) 1.4 (1.3) Refinement Resolution (Å) 46.0 – 3.5 50.0 – 2.4 No. reflections 65,915 347,924 R work / R free 36.35 / - 20.6/21.2 No. atoms Protein 5149 5192 Ligand/ion - - Water 0 196 B factors Protein 49.2 27.0 Ligand/ion - - Water - 30.6 r.m.s. deviations Bond lengths (Å) 0.011 0.013 Bond angles (°) 1.816 1.716 Open in a separate window a The data for O1M VP2 S93Y was collected from 14 different crystals b The data for A22 VP2 H83F was collected from 19 different crystals c Values in parentheses are for highest-resolution shell. caption a8 Data collection and refinement statistics Immunogenicity of stabilised mutants To compare the immunogenicity of wild-type and stabilised viruses, we vaccinated calves with purified inactivated wild-type or mutant (S93Y) virus of both the O1M and SAT2 serotypes.

Techniques: Staining, Purification, Generated

Journal: Nature structural & molecular biology

Article Title: Structure-based energetics of protein interfaces guide Foot-and-Mouth Disease virus vaccine design

doi: 10.1038/nsmb.3096

Figure Lengend Snippet: Capsid stabilising mutants for O1M, SAT2 and A22. A set of single amino acid substitutions at, or near the 2-fold axis, on the α-helix of the inter-pentamer interface is given in the top section. The change in binding free energy is calculated as described in Online Methods . Several mutants where found to be thermo- and pH- stable in three different serotypes of FMDV. A set of negative controls were also tested for O1M serotype to validate the simulation protocol. In one simulation VP2 arginine at position 60, that forms the salt bridge with the VP2 glutamate 212 on the opposite protomer, was substituted with either glycine or leucine; as expected, in both cases the interface was destabilised. Similarly, when glutamine at position 57, that makes a network of H-bonds across the interface, was substituted with glutamate or leucine an unstable interface was generated.

Article Snippet: The tyrosine density for O1M S93Y was also observed but it was less well defined due to the twinning of O1M crystals. ( e ) Cryo-EM maps showing the density for S93Y for O1M and SAT2 at 3.2Å and 3.5Å resolution, respectively. table ft1 table-wrap mode="anchored" t5 caption a7 O1M VP2 S93Y (a) A22 VP2 H93F (b) Data collection Space group I23 I222 Cell dimensions a , b , c (Å) 344.08, 344.08, 344.08 327.6, 341.3 363.6 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 46.0 – 3.5 (3.56 – 3.5) c 50.0 – 2.4 (2.49 - 2.4) R merge 0.46 0.27 I / σ I 1.7(0.76) 1.23 (0.5) Completeness (%) 77.7 (71.1) 45.5 (39.8) Redundancy 1.5(1.4) 1.4 (1.3) Refinement Resolution (Å) 46.0 – 3.5 50.0 – 2.4 No. reflections 65,915 347,924 R work / R free 36.35 / - 20.6/21.2 No. atoms Protein 5149 5192 Ligand/ion - - Water 0 196 B factors Protein 49.2 27.0 Ligand/ion - - Water - 30.6 r.m.s. deviations Bond lengths (Å) 0.011 0.013 Bond angles (°) 1.816 1.716 Open in a separate window a The data for O1M VP2 S93Y was collected from 14 different crystals b The data for A22 VP2 H83F was collected from 19 different crystals c Values in parentheses are for highest-resolution shell. caption a8 Data collection and refinement statistics Immunogenicity of stabilised mutants To compare the immunogenicity of wild-type and stabilised viruses, we vaccinated calves with purified inactivated wild-type or mutant (S93Y) virus of both the O1M and SAT2 serotypes.

Techniques: Binding Assay, Generated, Mutagenesis

Journal: Nature structural & molecular biology

Article Title: Structure-based energetics of protein interfaces guide Foot-and-Mouth Disease virus vaccine design

doi: 10.1038/nsmb.3096

Figure Lengend Snippet: (a) Plaques formed in BHK-21 cells by wild-type and mutant O1M and SAT2 viruses. The patterns of CPE correlated with plaque size. (b) Fluorescence assay to measure thermostability of infectious O1M viruses (left and middle panels) and inactivated SAT2 viruses (right panel) at pH 7.5 29. Wild-type O1M and SAT2 dissociated at 52.0°C and 47.0°C, respectively. Mutant S93Y dissociated at 53.5°C for both O1M and SAT2. O1M mutants S93F, S97Q and Y98F dissociated at 53.5°C, 54.0°C and 53.5°C, respectively. Mutant S93H (SAT2) dissociated at 51.0°C. S93W was similar to that of parental virus in both O1M and SAT2. (c) Thermo- and pH-stability of the recombinant empty capsids assayed by western blot after sedimentation through 15-45% sucrose density gradients. Dissociated capsids remain at the top of the gradient whereas intact capsids migrated near the bottom. Fractions were probed using anti-FMDV O1M polyclonal antibodies recognising VP139, 40 (see Supplementary Figure 3). Uncropped gels are shown in Supplementary Data Set 1.

Article Snippet: The tyrosine density for O1M S93Y was also observed but it was less well defined due to the twinning of O1M crystals. ( e ) Cryo-EM maps showing the density for S93Y for O1M and SAT2 at 3.2Å and 3.5Å resolution, respectively. table ft1 table-wrap mode="anchored" t5 caption a7 O1M VP2 S93Y (a) A22 VP2 H93F (b) Data collection Space group I23 I222 Cell dimensions a , b , c (Å) 344.08, 344.08, 344.08 327.6, 341.3 363.6 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 46.0 – 3.5 (3.56 – 3.5) c 50.0 – 2.4 (2.49 - 2.4) R merge 0.46 0.27 I / σ I 1.7(0.76) 1.23 (0.5) Completeness (%) 77.7 (71.1) 45.5 (39.8) Redundancy 1.5(1.4) 1.4 (1.3) Refinement Resolution (Å) 46.0 – 3.5 50.0 – 2.4 No. reflections 65,915 347,924 R work / R free 36.35 / - 20.6/21.2 No. atoms Protein 5149 5192 Ligand/ion - - Water 0 196 B factors Protein 49.2 27.0 Ligand/ion - - Water - 30.6 r.m.s. deviations Bond lengths (Å) 0.011 0.013 Bond angles (°) 1.816 1.716 Open in a separate window a The data for O1M VP2 S93Y was collected from 14 different crystals b The data for A22 VP2 H83F was collected from 19 different crystals c Values in parentheses are for highest-resolution shell. caption a8 Data collection and refinement statistics Immunogenicity of stabilised mutants To compare the immunogenicity of wild-type and stabilised viruses, we vaccinated calves with purified inactivated wild-type or mutant (S93Y) virus of both the O1M and SAT2 serotypes.

Techniques: Mutagenesis, Fluorescence, Recombinant, Western Blot, Sedimentation

Journal: Nature structural & molecular biology

Article Title: Structure-based energetics of protein interfaces guide Foot-and-Mouth Disease virus vaccine design

doi: 10.1038/nsmb.3096

Figure Lengend Snippet: The structures of engineered mutants O1M S93Y, A22 H93F and SAT2 S93Y were determined using X-ray crystallography and cryo-EM. (a) and (b) Stereo views of the density from cryo-EM reconstructions, the quality of the cryo-EM density maps allowed unambiguous fitting and refinement of the major capsid proteins for both (a) Cryo-EM structure of O1M S93Y at 3.2Å and (b) Cryo-EM structure of SAT2 S93Y at 3.5Å. (c) Predicted structures showing the stacking interactions of tyrosine and phenylalanine at position 93 of O1M VP2 proteins on the 2-fold symmetry axis. (d) X-ray structures of O1M S93Y and A22 H93F at 3.5Å and 2.4Å resolution, respectively. The electron-density map clearly showed a stacking interaction of phenylalanine side chains for A22 H93F. The tyrosine density for O1M S93Y was also observed but it was less well defined due to the twinning of O1M crystals. (e) Cryo-EM maps showing the density for S93Y for O1M and SAT2 at 3.2Å and 3.5Å resolution, respectively.

Article Snippet: The tyrosine density for O1M S93Y was also observed but it was less well defined due to the twinning of O1M crystals. ( e ) Cryo-EM maps showing the density for S93Y for O1M and SAT2 at 3.2Å and 3.5Å resolution, respectively. table ft1 table-wrap mode="anchored" t5 caption a7 O1M VP2 S93Y (a) A22 VP2 H93F (b) Data collection Space group I23 I222 Cell dimensions a , b , c (Å) 344.08, 344.08, 344.08 327.6, 341.3 363.6 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 46.0 – 3.5 (3.56 – 3.5) c 50.0 – 2.4 (2.49 - 2.4) R merge 0.46 0.27 I / σ I 1.7(0.76) 1.23 (0.5) Completeness (%) 77.7 (71.1) 45.5 (39.8) Redundancy 1.5(1.4) 1.4 (1.3) Refinement Resolution (Å) 46.0 – 3.5 50.0 – 2.4 No. reflections 65,915 347,924 R work / R free 36.35 / - 20.6/21.2 No. atoms Protein 5149 5192 Ligand/ion - - Water 0 196 B factors Protein 49.2 27.0 Ligand/ion - - Water - 30.6 r.m.s. deviations Bond lengths (Å) 0.011 0.013 Bond angles (°) 1.816 1.716 Open in a separate window a The data for O1M VP2 S93Y was collected from 14 different crystals b The data for A22 VP2 H83F was collected from 19 different crystals c Values in parentheses are for highest-resolution shell. caption a8 Data collection and refinement statistics Immunogenicity of stabilised mutants To compare the immunogenicity of wild-type and stabilised viruses, we vaccinated calves with purified inactivated wild-type or mutant (S93Y) virus of both the O1M and SAT2 serotypes.

Techniques: Cryo-EM Sample Prep

Journal: Nature structural & molecular biology

Article Title: Structure-based energetics of protein interfaces guide Foot-and-Mouth Disease virus vaccine design

doi: 10.1038/nsmb.3096

Figure Lengend Snippet: (a) Groups of four calves were vaccinated with purified inactivated virus of the O1M (left) and SAT2 (right) serotypes using in both cases either wild-type or mutant S93Y at days 0 and 28. The group mean virus neutralising antibody titre (VNT (log10)) are shown for blood samples assayed at 0, 7, 14, 21, 28, 35 and 42 days pv. Error bars represent the standard deviation. (b) Immunogenicity after long-term storage was assessed using inactivated wild-type and mutant S93Y FMDV SAT2 particles in a guinea pig trial. Two groups of 10 guinea pigs were each immunised with either mutant S93Y or wild-type SAT2 antigen. Prior to immunisation, formulated vaccines were stored for one month at 4°C. VNTs were assessed at three and six months pv. Error bars represent the standard deviation. (c) Equivalent aliquots of wild-type and stabilised SAT2 vaccines were stored for six months at 4°C prior to inoculation of two groups of ten guinea pigs. Only stabilised antigen shows evidence of protective VNTs. Error bars represent the standard deviation.

Article Snippet: The tyrosine density for O1M S93Y was also observed but it was less well defined due to the twinning of O1M crystals. ( e ) Cryo-EM maps showing the density for S93Y for O1M and SAT2 at 3.2Å and 3.5Å resolution, respectively. table ft1 table-wrap mode="anchored" t5 caption a7 O1M VP2 S93Y (a) A22 VP2 H93F (b) Data collection Space group I23 I222 Cell dimensions a , b , c (Å) 344.08, 344.08, 344.08 327.6, 341.3 363.6 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 46.0 – 3.5 (3.56 – 3.5) c 50.0 – 2.4 (2.49 - 2.4) R merge 0.46 0.27 I / σ I 1.7(0.76) 1.23 (0.5) Completeness (%) 77.7 (71.1) 45.5 (39.8) Redundancy 1.5(1.4) 1.4 (1.3) Refinement Resolution (Å) 46.0 – 3.5 50.0 – 2.4 No. reflections 65,915 347,924 R work / R free 36.35 / - 20.6/21.2 No. atoms Protein 5149 5192 Ligand/ion - - Water 0 196 B factors Protein 49.2 27.0 Ligand/ion - - Water - 30.6 r.m.s. deviations Bond lengths (Å) 0.011 0.013 Bond angles (°) 1.816 1.716 Open in a separate window a The data for O1M VP2 S93Y was collected from 14 different crystals b The data for A22 VP2 H83F was collected from 19 different crystals c Values in parentheses are for highest-resolution shell. caption a8 Data collection and refinement statistics Immunogenicity of stabilised mutants To compare the immunogenicity of wild-type and stabilised viruses, we vaccinated calves with purified inactivated wild-type or mutant (S93Y) virus of both the O1M and SAT2 serotypes.

Techniques: Purification, Mutagenesis, Standard Deviation