In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors

Figures

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  Fig. 1. (a) The overall structure of SV3CP with the β-strands labelled. The protein is composed of an N-terminal domain (red) containing a twisted 7-stranded antiparallel β-sheet and a C-terminal domain (green) consisting of a 6-stranded β-barrel. The catalytic triad of Cys139, His30 and Glu54 is shown in stick representation. (b) The putative tetrameric form of SV3CP. The asymmetric unit is composed of a dimer formed by chains denoted A and B. Crystallographic symmetry generates another identical dimer formed by chains denoted C and D. Both the AB and CD dimers have significant buried surface area suggesting that the dimer is physiologically significant. However another stable interface is formed between chains A and D and between chains B and C, resulting a tetrameric ensemble which may also have physiological significance. The domains of chain A are coloured and oriented the same as in (a) whereas chains B, C and D are coloured yellow, cyan and ochre. (c) Superimposition of the protein moieties of ligand-free SV3CP (green) and the polypeptide inhibitor-bound structure (Hussey et al., 2011; RCSB ID: 2iph) (pale brown). The ligand-free SV3CP structure has a shorter C-terminal end…

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  Fig. 2. Binding sites of the fragment screening hits. All the ligands bind in five sites labelled as A – E which are shown on a surface representation of the dimer. Site A coincides with the active site of the protease and site B is a putative RNA binding site. Site C coincides with the centre of the tetramer.

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  Fig. 3. Molecular structures of the fragment screening hits.

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  Fig. 4. The two active site binding fragments (site A). J01 (magenta) and J02 (green) are shown in (a) and (c) with the omit maps (contoured at 1.0 RMSD) coloured blue. The interactions between SV3CP and these fragments are shown in (b) and (d), respectively. Hydrogen bonds are indicated by dashed lines in cyan with the corresponding donor–acceptor atom distances shown in Å. Hydrophobic interactions are indicated by red eyebrow-like icons.

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  Fig. 5. A superposition of the two active-site binding fragment structures on a covalently bound substrate analogue inhibitor. The complexes with J01 and J02 are coloured magenta and green, respectively, while the polypeptide Michael acceptor inhibitor structure (RCSB ID: 2iph; Hussey et al., 2011) is shown in cyan. The C-terminal end of ligand-free SV3CP is labelled C1 and the corresponding part of the polypeptide inhibitor complex is labelled C2. The β-hairpin loop connecting strands β9 and β10 moves significantly from its position in the native structure (which is very close to its position in the J01 and J02 complexes) upon binding the polypeptide inhibitor.

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  Fig. 6. Interactions between the putative RNA binding site (site B) of the protease and fragments J03-J07. These are shown in 3D with the omit electron density contoured at 1.0 RMSD as (a, c, e, g, i) and in 2D with interacting residues shown in (b, d, f, h, j), respectively. Hydrogen bonds are indicated by dashed lines in cyan and hydrophobic interactions are indicated by red eyebrow-like icons. Protein chain identifiers are indicated by the letters A and B in brackets and those with a prime are from symmetry-related chains.

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  Fig. 7. Interactions between SV3CP and fragments J07-J17 which bind in site C at the centre of the putative tetramer. These are shown in 3D with the omit electron density contoured at 1.0 RMSD as (a, c, e, g, i, k, m, o, q, s, u) and in 2D with interacting residues shown in (b, d, f, h, j, l, n, p, r, t, v), respectively. Hydrogen bonds are indicated by dashed lines in cyan and hydrophobic interactions are indicated by red eyebrow-like icons. Protein chain identifiers are indicated by the letters A and B in brackets and those with a prime are from symmetry-related chains.

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  Fig. 8. Interactions made by fragments J18 and J19. The amide bond within J18 has apparently been cleaved and the resulting fragments, 2-ethyl-1,3,4-thiadiazole and trifluoroacetic acid, bind at sites D (a, b) and C (c, d), respectively. J19 binds at site E (e, f). These are shown in 3D with the omit electron density contoured at 1.0 RMSD. Hydrogen bonds are indicated by dashed lines in cyan and hydrophobic interactions are indicated by red eyebrow-like icons. Protein chain identifiers are indicated by the letters A and B in brackets and those with a prime are from symmetry-related chains.