An allosteric circuit in caspase-1

JOURNAL OF MOLECULAR BIOLOGY

卷:381

Article

Datta, Debajyoti^1,2  Scheer, Justin M.^1,2  Romanowski, Michael J.³  Wells, James A.^1,2 

[1] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA

[2] Univ Calif San Francisco, Dept Mol & Cellular Pharmacol, San Francisco, CA 94143 USA

[3] Sunesis Pharmaceut Inc, Dept Biol Struct, San Francisco, CA 94080 USA

关键词: caspase-1;    allostery;    alanine-scanning mutagenesis;    H-bonding;    cooperativity;   

DOI  :  10.1016/j.jmb.2008.06.040

来源: Elsevier

【 摘 要 】

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 similar to 15 angstrom 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 (k(cat)/K-m). 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 k(cat)/K-m. 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. (c) 2008 Elsevier Ltd. All rights reserved.

【 授权许可】

Free