An allosteric circuit in caspase-1 |
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Authors: | Datta Debajyoti Scheer Justin M Romanowski Michael J Wells James A |
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Affiliation: | 1 Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, CA 94143, USA 2 Department of Structural Biology, Sunesis Pharmaceuticals, Inc., South San Francisco, CA 94080, USA |
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Abstract: | Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface ∼ 15 Å from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them—Arg286 and Glu390, which form a salt bridge—have major effects, causing 100- to 200-fold reductions in catalytic efficiency (kcat/Km). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in kcat/Km. X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or “hot wire” that promotes site-to-site coupling. |
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Keywords: | ATCase, aspartate transcarbamoylase H-bonding, hydrogen bonding z-VAD-FMK, z-Val-Ala-Asp-fluoromethylketone PDB, Protein Data Bank Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Pipes, 1,4-piperazinediethanesulfonic acid PEG 2000 MME, polyethylene glycol 2000 monomethyl ether |
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