A common allosteric site and mechanism in caspases

Abstract

We present a common allosteric mechanism for control of inflammatory and apoptotic caspases. Highly specific thiol-containing inhibitors of the human inflammatory caspase-1 were identified by using disulfide trapping, a method for site-directed small-molecule discovery. These compounds became trapped by forming a disulfide bond with a cysteine residue in the cavity at the dimer interface Ϸ15 Å away from the active site. Mutational and structural analysis uncovered a linear circuit of functional residues that runs from one active site through the allosteric cavity and into the second active site. Kinetic analysis revealed robust positive cooperativity not seen in other endopeptidases. Recently, disulfide trapping identified a similar small-molecule site and allosteric transition in the apoptotic caspase-7 that shares only a 23% sequence identity with caspase-1. Together, these studies show a general small-molecule-binding site for functionally reversing the zymogen activation of caspases and suggest a common regulatory site for the allosteric control of inflammation and apoptosis.procaspase ͉ allosteric regulation ͉ apoptosis ͉ fragment-based discovery ͉ inflammation C aspases are dimeric thiol endopeptidases that cleave specific proteins after aspartic acid residues and drive apoptosis or inflammation (for review, see refs. 1 and 2). Although of considerable medical interest, the active sites of caspases have been very difficult to target with drug-like compounds owing to the enzyme's preference for negatively charged chemotypes with electrophilic warheads that engage the catalytic cysteine (3).In past work to develop alternative small-molecule inhibitors to the active site of the apoptotic caspase-7, we used a fragmentbased discovery approach called disulfide trapping. This is a site-directed small-molecule-capture approach in which natural or engineered cysteines on the surface of the protein are screened with a library of disulfide-containing small molecules in a redox buffer (4). This approach has been broadly applied in fragment-based drug discovery for both enzymes and challenging protein-protein interface targets (for review see ref. 5). Although the active-site cysteine of caspase-7 failed to yield strong hits from a library of Ϸ10,000 disulfide-containing compounds, several strong inhibitors were found for another cysteine in a cavity at the dimer interface.Procaspase-7, the inactive precursor or zymogen, is known to undergo a large structural transition after auto-or transproteolysis. This transition results in cleavage of the N-terminal 23 residues and an internal cut that yields two large and two small subunits of mature caspase-7. Structural studies of caspase-7 containing the disulfide-trapped inhibitors at the dimerinterface cavity showed that the compounds reversed this structural transition and induced a conformation virtually identical to the zymogen form (6).We wondered whether other caspases could be inhibited by a similar mechanism. The class of human inf lammator y caspases-1, -4, and -5 is...

Mechanism of procaspase-8 activation by c-FLIP L

“…5A). Recent studies of caspase-1 and caspase-7 revealed that compounds targeting the dimerization interface, Ϸ15 Å away from the active site, can propagate structural changes that ultimately deform the active sites (19,20). Perhaps the most convincing observation from our work in support of this conjecture emerges from the fact that the ␤6 interface is the most conserved element among all of the regions of contact between FLIP and z-C8 (Fig.…”

Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog

“…In addition, our data suggest new approaches for inhibiting AdoMetDC in trypanosomes, either by blocking formation of the AdoMetDC-prozyme complex and/or by stabilizing the inactive conformation of AdoMetDC. Successful strategies for these approaches have recently been described for other proteins (29)(30)(31).…”

Zinc-mediated Allosteric Inhibition of Caspase-6

“…We have previously observed how phosphorylation of caspase-6 at Ser-257 results in a steric clash of as few as three atoms in the side chain of Pro-201, which leads to allosteric inhibition by preventing the active conformation of one loop. In caspase-7 (45,63,64) or -1 (65,66), binding small synthetic allosteric inhibitors at the dimer interface leads to a series of side chain conformational changes that ultimately block the substrate-binding groove, preventing proteolytic activity. These examples and a great number of other examples from a large number of proteins are satisfying as it is possible to visualize the series of side chain conformational changes that lead to inactivation.…”