|Jeanne A. Hardy
Associate Professor of Chemistry, University of Massachusetts
Ph.D.: University of California at Berkeley
Design of allosteric pathways in caspases and phosphatases; x-ray crystallography
Many biological responses are triggered when small chemical moieties bind to cavities or surfaces of proteins. Attention typically focuses on small molecules binding to the main functional site, which is termed the protein active site. In other cases, binding of small molecules to external sites, called allosteric sites, dramatically influences the activity of the protein. Some well known allosteric sites like those in hemoglobin, citrate synthase and glycogen phosphorylase were discovered many years ago; however, new allosteric sites like the one in caspase-7 (right, red) are being uncovered serendipitously all the time. We aim to understand and exploit allosteric regulation by designing allosteric binding sites for alternative small molecules. We redesign both known and novel allosteric sites to be controlled by the small molecule of our choice. In the figure at the right a novel allosteric site cavity in caspase-7 (red) is occupied by a small molecule we chose based on its size, solubility and exceptional drug-like properties (yellow sticks). Redesigning this novel allosteric site to bind to an alternative small molecule will allow us to regulate caspase-7 activity independently of all other caspases.
Designed allosteric proteins are of great utility because of their exquisite innate specificity. Very often new small molecules that interact with protein active or allosteric sites have a broad specificity because identical sites exist on other members of that family of proteins. The diagram at the left illustrates the situation where all of the family members have identically shaped active sites. Thus active-site inhibitors typically interact with all the members of a family of proteins. Designing allosteric sites to bind to the small molecule we choose allows that small molecule to serve as an exquisitely specific switch to turn on or turn off activity of the designed allosteric protein independent of any related family members. Because none of the wild-type family members contain the designed site, they have no affinity for the designed inhibitor, and thus are unaffected by the presence of the novel inhibitor.
We use computational protein design methods to engineer new allosteric sites, molecular biology to introduce these sites into proteins of interest, biochemical and cell-based assays to determine the inhibitory ability of our designed small molecule-protein pair, and x-ray crystallography to determine that our designed mode of binding occurs. Once we are convinced that we have designed an optimal pair, we use the small molecule as a chemical genetics agent in any relevant biological situation.
In principle, designed allosteric sites can be engineered into proteins in any family in any biological system. Apoptosis (programmed cell death) is a fascinating biological system of great therapeutic importance. Blocking apoptosis is a useful way of treating cells that are routed to death following ischemic events such as stroke or heart attack or in long-term diseases such as Alzheimer's Disease. Activating apoptosis is the best known way to induce proliferating cancer cells to die, and many of the best known cancer therapies work via this mechanism. While a great deal of research effort has focused on the regulation of apoptosis, many important details remain to be discovered. Designed allosteric sites will allow us to understand the precise roles of caspases, phosphatases and other large families of proteins involved in apoptosis. This understanding will lead us to discover new drug targets and give us new methods of controlling apoptosis and treating disease.
Velazquez-Delgado, E. M. and Hardy, J. A., 2012. “Zinc-Mediated Allosteric Inhibition of Caspase-6.”Journal of Biological Chemistry. 287(43), 36000.
Huber, K.L. and Hardy J.A., 2012 “Mechanism of zinc-mediated inhibition of caspase-9.” Protein Science. 21,1056-1065.. Article featured as cover illustration.
Huber, K.L., Ghosh, S. and Hardy J.A., 2012. “Inhibition of caspase-9 by stabilized peptides targeting the dimerization interface.”Peptide Science. 98(5), 451–465.
Velazquez-Delgado, E. M. and Hardy J.A., 2012. “Phosphorylation regulates assembly of the caspase-6 substrate-binding groove.” Structure. 20, 742–751.
Vaidya S., Hardy J.A., Caspase-6 Latent State Stability Relies on Helical Propensity.
Vaidya S., Velázquez-Delgado E.M., Abbruzzese G., Hardy J.A. Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6. Journal of Molecular Biology 2011, 406(1):75-91.
Witkowski, W.A., Hardy, J.A. "L2 loop is critical for caspase-7 active site formation" Protein Science, 2009, 18, 1459-1468. Article featured as cover illustration.
Hardy, J. A. and Wells, J. “Dissecting an Allosteric Switch in Caspase-7 using Chemical and Mutational Probes.” Journal Biological Chemistry 2009, 284(38):26063-9.
Hardy, J. A., Lam, J., Nguyen, J. T., O’Brien, T. and Wells, J. “Discovery of an allosteric site in caspases.” Proc. Natl. Acad. Sci. U S A. 2004, 101(34), 12461-6.
Protein Design Papers:
Wu, P., Nicholls, S., and Hardy, J.A. 2013 “A tunable, modular approach to fluorescent protease-activated reporters.” in Press Biophysical Journal.
Nicholls, S. and Hardy, J.A. 2013 “Structural Basis of Fluorescence Quenching in Caspase Activatable-GFP.” Protein Science. 22(3), 247. Article featured as cover illustration.
López, G.E, Colón-Díaz, I., Cruz, A., Ghosh,S., Nicholls, S.B., Viswanathan, U., Hardy, J.A. and Auerbach, S.M. 2012 “Modeling non-aqueous proton wires tethered to helical peptides: Biased proton transfer driven by helical dipoles.” The Journal of Physical Chemistry A. 116(4):1283-8.
Nicholls, S., Chu, J., Abbruzzese, G. Tremblay, K.D. and Hardy, J.A. “Mechanism of a dark-to- bright reporter of caspase activity.”Journal of Biological Chemistry, 286 (28), 24977-24986.
Witkowski, W. and Hardy, J.A. “A designed redox-controlled caspase”Protein Science, 20, 1421-1431. Article featured as cover illustration.
Huber, K.L., Olson, K.D. and Hardy, J.A. "Robust production of a peptide library using methodological synchronization" Protein Expression and Purification. 2009, 67,139-147.
Hardy, J. A. and Nelson, H. C. M. “Proline in an alpha-helical kink is required for folding kinetics but not for kinked structure, function or stability of heat shock transcription factor” Protein Science, 2000, 9(11), 2128-2141.
Hardy, J. A., Walsh, S. T. R., and Nelson, H. C. M. “Role of an alpha-Helical Bulge in the Yeast Heat Shock Transcription Factor” J. Mol. Biol., 2000, 295(3), 393-409.
Protein Design Papers:
Hardy, J. A. and Wells, J. “Searching for Allosteric Sites in Enzymes.” Curr. Opin. Str. Biol. 2004, 14(6), 706-715.
Hardy, J.A. "Discovery and exploitation of allosteric sites for control of protein function." Nanomedicine: Nanotechnology, Biology and Medicine 2006, 2(4), 291.
Hardy, J. A. “A Link Means a Lot: Disulfide Tethering in Structure-Based Drug Design” in Computational Approaches to Structure Based Drug Design, publishers Royal Society of Chemistry, 2007, 318-347.