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| - NMR Solution Structures of the Apo and Peptide-Inhibited Human Rhinovirus 3CProtease (Serotype 14): Structural and Dynamic Comparison
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| - The human rhinovirus (HRV) is a positive sense RNA virus responsible for about 30% of“common colds”. It relies on a 182 residue cysteine protease (3C) to proteolytically process its singlegene product. Inhibition of this enzyme in vitro and in vivo has consistently demonstrated cessation ofviral replication. This suggests that 3C protease inhibitors could serve as good drug candidates. However,significant proteolytic substrate diversity exists within the 110+ known rhinovirus serotypes. To investigatethis variability we used NMR to solve the structure of the rhinovirus serotype 14 3C protease (subgenusB) covalently bound to a peptide (acetyl-LEALFQ-ethylpropionate) inhibitor. The inhibitor-bound structurewas determined to an overall rmsd of 0.82 Å (backbone atoms) and 1.49 Å (all heavy atoms). Comparisonwith the X-ray structure of the serotype 2 HRV 3C protease from subgenus A (51% sequence identity)bound to the inhibitor ruprintrivir allowed the identification of conserved intermolecular interactionsinvolved in proximal substrate binding as well as subgenus differences that might account for the variabilityobserved in SAR studies. To better characterize the 3C protease and investigate the structural and dynamicdifferences between the apo and bound states we also solved the solution structure of the apo form. Theapo structure has an overall rmsd of 1.07 ± 0.17 Å over backbone atoms, which is greater by 0.25 Å thanwhat is seen for the inhibited enzyme (2B0F.pdb). This increase is localized to the enzyme's C-terminalβ-barrel domain, which is responsible for recognizing and binding proteolytic substrates. Amide hydrogenexchange dynamics revealed dramatic differences between the two enzyme states. Furthermore, a numberof residues exhibited exchange-broadened amide NMR signals in the apo state compared to the inhibitedstate. The majority of these residues are associated with proteolytic substrate interaction.
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