An Allosteric Circuit in Caspase-1

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 ( 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.