Allostery in Caspases
- Author(s): Datta, Debajyoti;
- Advisor(s): Wells, James A;
- et al.
Caspases are a family of dimeric thiol proteases central to the inflammatory and apoptotic pathways. Structural studies of the major inflammatory caspase, caspase-1, reveal that the enzyme exists in two states: an on-state when the active site is occupied; or an off-state when the active site is empty or when the enzyme is bound by allosteric ligand at the dimer interface. Determining which residues and regions of the enzyme are responsible for determining enzyme conformation and for regulating transitions between states would elucidate the molecular basis for allostery in caspase-1.
A network of 21 hydrogen bonds from 9 side chains connecting the active and allosteric sites change partners when going between the on- and off-states. Alanine-scanning mutagenesis of these 9 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). More detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: a homologous R286K substitution causes a 150-fold reduction in kcat/KM. Thus, side chains from only a small subset of the larger H-bonding network are 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 on to the second active site. This subset constitutes a functional allosteric circuit or "hot-wire" that promotes site-to-site coupling.
Further experiments demonstrate the utility of using site-directed mutagenesis and chemical ligands to select for and examine enzyme conformation. Targeting specific glycine and proline residues in caspase-1 for mutation selects for different conformational states of the enzyme. We present a method of using covalent inhibitors for caspases to create half-labeled hybrid constructs that trap the conformational state of caspases in solution and allow us to understand how allostery plays a role in caspase activity. We present a model that provides a mechanism by which the very conformational state of the various caspase family members recapitulates their role in specific biologic pathways and provides an additional level of regulation.