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Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.

ABSTRACT: Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.

SUBMITTER: Tang Y

PROVIDER: S-EPMC3190824 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.

Tang Yinyan Y Wells James A JA Arkin Michelle R MR

The Journal of biological chemistry 20110802 39

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined ...[more]

PMID: 21828056

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Project description:Hexachlorobenzene (HCB), as one of the persistent organic pollutants (POPs) and a possible human carcinogen, is especially resistant to biodegradation. In this study, HcbA1A3, a distinct flavin-N5-peroxide-utilizing enzyme and the sole known naturally occurring aerobic HCB dechlorinase, was biochemically characterized. Its apparent preference for HCB in binding affinity revealed that HcbA1 could oxidize only HCB rather than less-chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with flavin mononucleotide (FMN) were resolved, revealing HcbA1 to be a new member of the bacterial luciferase-like family. A much smaller substrate-binding pocket of HcbA1 than is seen with its close homologues suggests a requirement of limited space for catalysis. In the active center, Tyr362 and Asp315 are necessary in maintaining the normal conformation of HcbA1, while Arg311, Arg314, Phe10, Val59, and Met12 are pivotal for the substrate affinity. They are supposed to place HCB at a productive orientation through multiple interactions. His17, with its close contact with the site of oxidation of HCB, probably fixes the target chlorine atom and stabilizes reaction intermediates. The enzymatic characteristics and crystal structures reported here provide new insights into the substrate specificity and catalytic mechanism of HcbA1, which paves the way for its rational engineering and application in the bioremediation of HCB-polluted environments.IMPORTANCE As an endocrine disrupter and possible carcinogen to human beings, hexachlorobenzene (HCB) is especially resistant to biodegradation, largely due to difficulty in its dechlorination. The lack of knowledge of HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase, HcbA1A3, representing the only naturally occurring aerobic HCB dechlorinase known so far, was reported. Here, we report its biochemical and structural characterization, providing new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide-utilizing enzymes.

Dataset Information

Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.

Publications

Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.

Similar Datasets

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