Faculty Profile: Edward Hawrot, Ph.D., Harvard University, 1976

Edward Hawrot
Edward Hawrot, Ph.D., Harvard University, 1976
Alva O. Way University Professor of Medical Science
Molecular Pharmacology, Physiology & Biotechnology
Work: +1 401-863-1034
We pursue biochemical and pharmacological studies aimed at understanding the fundamental structure-function relationship of nicotinic acetylcholine receptors (nAChRs). We also study the molecular basis for the highly specific recognition of muscle-type nAChRs by certain snake venom-derived toxins classified as alpha-neurotoxins. More recently, we have used homologous recombination techniques to construct a knock-in mouse in which the alpha3 gene encoding one subtype of neuronal nAChRs has been minimally mutated to impart pharmacological sensitivity to the classic nicotinic antagonist, alpha-bungarotoxin. These mice should enable a systematic determination of the role of alpha3-containing nAChRs in behavior and nervous system function.

Biography

I received my Ph.D. in Biochemistry from Harvard and then moved from Gene Kennedy's lab in Biological Chemistry to the Department of Neurobiology at Harvard Medical School as a Helen Hay Whitney Postdoctoral Fellow working with Paul Patterson. Subsequently, after ten years on the faculty in the Department of Pharmacology at Yale University School of Medicine, I relocated to Brown University in 1990 initially as Chair of the Section of Molecular Pharmacology in the Division of Biology and Medicine. As a past Established Investigator of the American Heart Association and Upjohn Professor of Pharmacology, my research interests include the understanding of the structure and function of nicotinic acetylcholine receptors and of the neurotoxins that target these important receptors.

Institutions

Bu

Research Description

The nicotinic acetylcholine receptor (nAChR) mediates synaptic transmission at the neuromuscular junction, in peripheral autonomic ganglia, and in the central nervous system. The nAChR belongs to a super-family of ligand-gated channels which include the glycine and GABA-A receptors. In the case of the nAChR, receptor activation occurs after binding of the physiological agonist, acetylcholine (ACh), to the receptor complex leading to the transient formation of a cation-selective channel. Antagonists with relative specificity for the nAChR include tubocurarine and the snake venom-derived neurotoxins such as alpha-bungarotoxin (BGTX) and alpha-cobratoxin. Our major interest is in determining the molecular and structural basis for the specificity of drug-receptor interactions. To accomplish this we are carrying out protein and peptide biophysical studies of receptor fragments. These studies include determining the solution structure of toxin-receptor fragment complexes using modern multidimensional NMR spectroscopic techniques. We also are applying molecular biological approaches to this problem by using heterologous expression and site-directed mutagenesis of both receptor and toxin residues in order to elucidate the structure-function relationship in this system. Both approaches are complementary and will facilitate the complete structural determination of the ligand binding site in the nicotinic acetylcholine receptor. Such information could provide the basis for the rational design of drugs selective for such sites.

More recent efforts are directed to exploiting the binding determinants responsible for BGTX action by transplanting these into other subunits normally insensitive to BGTX.  We have termed the peptide sequence with this property a "pharmatope".  We find that in some cases BGTX binding to such a pharmatope produces functional blockade of receptor action through an allosteric as opposed to a competitive mechanism.  One further advantage to a pharmatope-tagged receptor subunit is that the many commercially available derivates of BGTX can now be used to label and functionally inhibit receptor complexes containing the tagged subunit.  We are now pushing this approach to the organismic level by preparing a mouse using the homologous recombination technique (i.e., knock-in) in which the alpha3 gene is replaced by one containing a five amino substitution rendering the mutant subunit sensitive to BGTX, whereas the wild-type alpha3 is completely insensitive to BGTX.  This mouse was the pioneer mouse generated in the Brown University Transgenic Facility in collaboration with Dr. Jan Klysik.  The heterozygous mice are completely normal and breed well.  We are currently assessing the functional sensitivity of the neuronal nicotinic receptors in the peripheral ganglia from these knock-in mice.  The aim here is to use these mice to explore the physiological function of the alpha3 subunit in the central nervous system utilizing a standard pharmacological dissection approach.

Many drugs—both those that are therapeutically useful and those that are not—physically interact with receptor proteins found on the cell surface of neurons and other excitable tissue. I am interested in determining the molecular and structural basis for the remarkable specificity of drug-receptor interactions in the brain. In particular, I am interested in the nicotinic acetylcholine receptor, which mediates synaptic transmission at the junction between motor nerves and skeletal muscle. The function of the neuromuscular junction can be completely blocked by alpha-neurotoxins derived from snake venom that work by binding to the receptor. The specificity and potency of the alpha-neurotoxins found in snake venom are so great that they represent an ideal toward which drug developers and medicinal chemists strive. The ideal is a drug extremely active at low concentrations that does not interact with irrelevant receptors, thus reducing the likelihood of side effects. Working toward that ideal, and to increase our understanding of how receptors work, I am pursuing a number of biophysical studies with other researchers involving receptor fragments. With more detailed structural information describing the toxin-receptor interface, it should be possible for us to engineer the toxin by mutagenesis so as to alter its specificity.

I became interested in this line of research as a result of my interests in chemistry and in the functioning of the brain. Both of these areas of interest were satisfied by studies of drugs and toxins and their interactions with receptors important for brain and nervous system function.

Grants and Awards

2005-2009 NIH (CSR) BSCT/BPNS Study Section (Biophysics of Neural Systems)
2010 NIH (CSR) ZRG1 MDCN-B(02) Special Emphasis Panel
2011 NIH (CSR) MNPS Study Section (ad hoc member)
2013 NIH (CSR) NINDS SNRP Study Section (ad hoc)

2011 Fellow of the American Association for the Advancement of Science (AAAS)

Affiliations

2001-2006 Editorial Board, Journal of Biological Chemistry

Member, American Association for the Advancement of Science, American Society for Pharmacology & Experimental Therapeutics, American Society for Biochemistry and Molecular Biology, Society for Neuroscience, Association of Medical School Pharmacology Chairs

Funded Research

2010-2011 NIH/NCRR (1S10RR027027-01) Acquisition of an LTQ Orbitrap Velos ETD Mass Spectrometer

2010-2015 NIH (1T32GM077995-01A2) Predoctoral Training Program in Trans-Disciplinary Pharmacological Sciences

2010-2012 National Science Foundation (EPS-1005789) High-Capacity Cyber-Connectivity to the Jewelry District Campuses in Providence, RI

2010-2015 National Science Foundation (EPS-10040507; subaward to Brown University from URI) Rhode Island Experimental Program to Stimulate Competitive Research (RI-EPSCoR): Infrastructure to Advance Life Sciences in the Ocean State

2011-2013 NIH/NIA (1R21AG038774-01A1) Effects of Alzheimer's Disease on Hippocampal alpha7-nAChR Protein Interactors

Selected Publications

  • Peng, C., Chen, W., Sanders, T., Chew, G., Liu, J., Hawrot, E., and Chi, C., Chemical Synthesis and Characterization of Two αlpha4/7-Conotoxins. Epub 2010 Aug 27, Acta Biochim Biophys Sinica (Shanghai), Oct;42(10):745-753, 2010. (2010)
  • Peng, C., Ye, M., Wang, Y., Shao, X., Yuan, D., Jing Liu, J., Hawrot, E., Wang, C., Chi, C., A New Subfamily of Conotoxins Belonging to the A-Superfamily. Epub August 4, 2010, Peptides 31:2009-2016, 2010. (2010)
  • Moise, L., Liu, J., Pryazhnikov, E., Khiroug, L., Jeromin, A., and Hawrot, E. (2010) Kv4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization. Channels (Austin), 4(2):115-123. Epub 2010 March 8, PMCID: PMC2888848. (2010)
  • Peng, C., Chen, W., Han, Y., Sanders, T., Chew, G., Liu, J., Hawrot, E., Chi, C., and Wang, C., (2009) Characterization of a novel alpha4/4-conotoxin, Qc1.2, from vermivorous Conus quercinus. Acta Biochim Biophys Sinica (Shanghai), 41: 858-864. (2009)
  • Caffery, P.M., Krishnaswamy, A., Sanders, T., Liu, J., Hartlaub, H., Klysik, J., Cooper, E., and Hawrot, E. (2009) Engineering neuronal nicotinic acetylcholine receptors with functional sensitivity to alpha-bungarotoxin: A novel alpha3-knock-in mouse. Eur. J. Neurosci., 30:2064-2076, PMCID: PMC2818262. (2009)
  • Paulo, J.A., Brucker, W.J., and Hawrot, E., (2009) Proteomic analysis of an alpha7 nicotinic acetylcholine receptor interactome. J. Proteome Research 8: 1849-1858, PMCID: PMC289157. (2009)
  • Paulo, J.A. and Hawrot, E., (2009) Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity. Protein Expression and Purification, 67: 76-81, PMCID: PMC2713376. (2009)
  • Paulo, J.A. and Hawrot, E., (2009) A radio-isotope label-free alpha-bungarotoxin binding assay using Biacore sensor chip technology for real-time analysis. Analytical Biochemistry 389: 86-88, PMCID: PMC2684811. (2009)
  • Goldman, R.E., Kingdon, C., Wasser, J., Clark, M.A., Goldberg, R., Papandonatos, G.D., Hawrot, E., and Koren, G. (2008) Rhode Islanders' Attitudes towards the Development of a Statewide Genetic Biobank. Personalized Medicine 5: 339-359. (2008)
  • Peng, C., Han, Y., Sanders, T., Chew, G., Liu, J., Hawrot, E., and Wang, C. (2008) Alpha4/7-conotoxin Lp1.1 is a Novel Antagonist of Neuronal Nicotinic Acetylcholine Receptors. Peptides 29:1700-1707. (2008)
  • Liu, L., Chew, G., Hawrot, E., Chi, C., and Wang, C. Two Potent Alpha3/5 Conotoxins from Piscivorous Conus achatinus. Acta Biochimica et Biophysica Sinica 39: 438-444, 2007. (2007)
  • Sanders, T. and Hawrot, E. (2004) A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors. Journal of Biological Chemistry; 279:51460-5. http://www.jbc.org/cgi/content/full/279/49/51460 (2004)
  • Moise, L., Zeng, H., Caffery, P., Rogowski, R.S., and Hawrot, E. (2002) Structure and function of alpha-bungarotoxin. Journal of Toxicology-Toxin Reviews, 21(3):293-317. http://search.epnet.com/direct.asp?an=7705507&db=aph (2002)
  • Zeng, H. and Hawrot, E. (2002) NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha1 subunit of the nicotinic acetylcholine receptor from Torpedo californica. Journal of Biological Chemistry 277:37439-37445. http://www.jbc.org/cgi/content/full/277/40/37439 (2002)
  • Moise, L., Piserchio, A., Basus, V.J., Hawrot, E. (2002) NMR structural analysis of alpha-bungarotoxin and its complex with the principal alpha-neurotoxin binding sequence on the alpha7 subunit of a neuronal nicotinic acetylcholine receptor. Journal of Biological Chemistry 277:12406-17. http://www.jbc.org/cgi/content/full/277/14/12406 (2002)
  • Zeng, H., Moise, L., Grant, M.A., & Hawrot, E. (2001) The solution structure of the complex formed between alpha-bungarotoxin and an 18mer cognate peptide derived from the alpha1 subunit of the nicotinic acetylcholine receptor from Torpedo californica. Journal of Biological Chemistry, 276:22930-40. (http://www.jbc.org/cgi/content/full/276/25/22930) (2001)
  • Blein, S., Hawrot, E., & Barlow, P. (2000) The metabotropic GABA receptor: molecular insights and their functional consequences. Cell and Molecular Life Science, 57: 635-650. (2000)
  • Levandoski, M.M., Caffery, P., Rogowski, R.S., Shi, Q.-L., & Hawrot, E. (2000) Recombinant expression of alpha-bungarotoxin in Pichia pastoris facilitates identification of mutant toxins engineered to recognize neuronal nicotinic acetylcholine receptors. The Journal of Neurochemistry, 74: 1279-1289. (2000)
  • Spura, A., Riel, R., Freedman, N.D., Agrawal, S., Seto, C., & Hawrot, E. (2000) Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding modes for alpha-bungarotoxin and Erabutoxin a. Journal of Biological Chemistry 275: 22452-22460. (http://www.jbc.org/cgi/content/full/275/29/22452) (2000)
  • Levandoski, M.M., Lin, Y., Moise, L., McLaughlin, J.T., Cooper, E., & Hawrot, E. (1999) Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin. Journal of Biological Chemistry 274: 26113-26119. (1999)
  • Grant, M.A., Gentile, L.N., Shi, Q.-L., Pellegrini, M., and Hawrot, E. (1999) Expression and spectroscopic analysis of soluble nicotinic acetylcholine receptor fragments derived from the extracellular domain of the alpha-subunit. Biochemistry 38: 10730-10742. (1999)
  • Rosenthal, J.A., Levandoski, M.M., Chang, B., Potts, J.F., Shi, Q.-L., and Hawrot, E. (1999) The functional role of positively charged amino acid side chains in alpha-bungarotoxin revealed by site-directed mutagenesis of a His-tagged recombinant alpha-bungarotoxin. Biochemistry 38: 7847-7855. (1999)
  • Spura, A., Russin, T.S., Freedman, N., Grant, M., McLaughlin, J.T., & Hawrot, E. (1999) Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site. Biochemistry 38: 4912-4921. (1999)
  • Hawrot, E., Xiao, Y., Shi, Q.-L., Norman, D., Kirkitadze, M., and Barlow, P. (1998) Demonstration of a tandem pair of complement protein modules in GABA-B receptor 1a. FEBS Letters 432: 103-108. (1998)