Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

Logo for

PNAS 101 (34): 12461-12466

Copyright © 2004 by the National Academy of Sciences.

BIOCHEMISTRY

Discovery of an allosteric site in the caspases

Jeanne A. Hardy, Joni Lam, Jack T. Nguyen {dagger}, Tom O'Brien, and James A. Wells {ddagger}

Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco, CA 94080

Communicated by Robert M. Stroud, University of California, San Francisco, CA, July 2, 2004

Received for publication May 11, 2004.

Abstract: Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 Å from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.

Freely available online through the PNAS open access option.

Abbreviations: DICA, 2-(2,4-dichloro-phenoxy)-N-(2-mercapto-ethyl)-acetamide; FICA, 5-fluoro-1H-indole-2-carboxylic acid (2-mercapto-ethyl)-amide; DEVD, Asp-Glu-Val-Asp.

Data deposition: The atomic coordinates of the DICA and FICA complexes with caspase-7 have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1SHJ and 1SHL, respectively).

{dagger} Present address: Catalyst Biosciences, Inc., 225 Gateway Boulevard, South San Francisco, CA 94080.

The term Tethering is a service mark of Sunesis Pharmaceuticals for its fragment-based drug discovery method.

{ddagger} To whom correspondence should be addressed. E-mail: jaw{at}sunesis.com.

© 2004 by The National Academy of Sciences of the USA

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Structural snapshots reveal distinct mechanisms of procaspase-3 and -7 activation.
N. D. Thomsen, J. T. Koerber, and J. A. Wells (2013)
PNAS 110, 8477-8482
   Abstract »    Full Text »    PDF »
Zinc-mediated Allosteric Inhibition of Caspase-6.
E. M. Velazquez-Delgado and J. A. Hardy (2012)
J. Biol. Chem. 287, 36000-36011
   Abstract »    Full Text »    PDF »
Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation.
J. A. Zorn, D. W. Wolan, N. J. Agard, and J. A. Wells (2012)
J. Biol. Chem. 287, 33781-33795
   Abstract »    Full Text »    PDF »
Structure-based model of allostery predicts coupling between distant sites.
P. Weinkam, J. Pons, and A. Sali (2012)
PNAS 109, 4875-4880
   Abstract »    Full Text »    PDF »
Turning a protein kinase on or off from a single allosteric site via disulfide trapping.
J. D. Sadowsky, M. A. Burlingame, D. W. Wolan, C. L. McClendon, M. P. Jacobson, and J. A. Wells (2011)
PNAS 108, 6056-6061
   Abstract »    Full Text »    PDF »
A geometry-based generic predictor for catalytic and allosteric sites.
S. Mitternacht and I. N. Berezovsky (2011)
Protein Eng. Des. Sel. 24, 405-409
   Abstract »    Full Text »    PDF »
Allostery Is an Intrinsic Property of the Protease Domain of DegS: IMPLICATIONS FOR ENZYME FUNCTION AND EVOLUTION.
J. Sohn, R. A. Grant, and R. T. Sauer (2010)
J. Biol. Chem. 285, 34039-34047
   Abstract »    Full Text »    PDF »
Caspase-7 Cleavage of Kaposi Sarcoma-associated Herpesvirus ORF57 Confers a Cellular Function against Viral Lytic Gene Expression.
V. Majerciak, M. Kruhlak, P. K. Dagur, J. P. McCoy Jr, and Z. M. Zheng (2010)
J. Biol. Chem. 285, 11297-11307
   Abstract »    Full Text »    PDF »
Small-Molecule Activators of a Proenzyme.
D. W. Wolan, J. A. Zorn, D. C. Gray, and J. A. Wells (2009)
Science 326, 853-858
   Abstract »    Full Text »    PDF »
Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes.
J. A. Hardy and J. A. Wells (2009)
J. Biol. Chem. 284, 26063-26069
   Abstract »    Full Text »    PDF »
Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A.
R. N. Pruitt, B. Chagot, M. Cover, W. J. Chazin, B. Spiller, and D. B. Lacy (2009)
J. Biol. Chem. 284, 21934-21940
   Abstract »    Full Text »    PDF »
Mechanism of procaspase-8 activation by c-FLIPL.
J. W. Yu, P. D. Jeffrey, and Y. Shi (2009)
PNAS 106, 8169-8174
   Abstract »    Full Text »    PDF »
Crystal Structure of Procaspase-1 Zymogen Domain Reveals Insight into Inflammatory Caspase Autoactivation.
J. M. Elliott, L. Rouge, C. Wiesmann, and J. M. Scheer (2009)
J. Biol. Chem. 284, 6546-6553
   Abstract »    Full Text »    PDF »
A Novel Mode of Intervention with Serine Protease Activity: TARGETING ZYMOGEN ACTIVATION.
G. E. Blouse, K. A. Botkjaer, E. Deryugina, A. A. Byszuk, J. M. Jensen, K. K. Mortensen, J. P. Quigley, and P. A. Andreasen (2009)
J. Biol. Chem. 284, 4647-4657
   Abstract »    Full Text »    PDF »
Triggering Protein Folding within the GroEL-GroES Complex.
D. Madan, Z. Lin, and H. S. Rye (2008)
J. Biol. Chem. 283, 32003-32013
   Abstract »    Full Text »    PDF »
Isoquinoline-1,3,4-trione Derivatives Inactivate Caspase-3 by Generation of Reactive Oxygen Species.
J.-Q. Du, J. Wu, H.-J. Zhang, Y.-H. Zhang, B.-Y. Qiu, F. Wu, Y.-H. Chen, J.-Y. Li, F.-J. Nan, J.-P. Ding, et al. (2008)
J. Biol. Chem. 283, 30205-30215
   Abstract »    Full Text »    PDF »
Small Molecule-Induced Allosteric Activation of the Vibrio cholerae RTX Cysteine Protease Domain.
P. J. Lupardus, A. Shen, M. Bogyo, and K. C. Garcia (2008)
Science 322, 265-268
   Abstract »    Full Text »    PDF »
Proteolytic Cleavage of Ataxin-7 by Caspase-7 Modulates Cellular Toxicity and Transcriptional Dysregulation.
J. E. Young, L. Gouw, S. Propp, B. L. Sopher, J. Taylor, A. Lin, E. Hermel, A. Logvinova, S. F. Chen, S. Chen, et al. (2007)
J. Biol. Chem. 282, 30150-30160
   Abstract »    Full Text »    PDF »
Whence cometh the allosterome?.
J. E. Lindsley and J. Rutter (2006)
PNAS 103, 10533-10535
   Abstract »    Full Text »    PDF »
A common allosteric site and mechanism in caspases.
J. M. Scheer, M. J. Romanowski, and J. A. Wells (2006)
PNAS 103, 7595-7600
   Abstract »    Full Text »    PDF »
A viral caspase contributes to modified apoptosis for virus transmission.
D. K. Bideshi, Y. Tan, Y. Bigot, and B. A. Federici (2005)
Genes & Dev. 19, 1416-1421
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882