Ph.D. 1993, Weizmann Inst.; Sen. Lect. 1997
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Structural determination of biologically related macromolecules via X-ray crystallographic techniques.
Ligand recognition, initial signaling events, and the role of dimer/oligomer orientation in cytokine receptor systems.
Structural studies of avidin/streptavidin - biotin high affinity systems.
Structural based drug design and optimization.
Combinatorial approaches in drug discovery.
Abstracts of Current Research:
Signaling attenuation in cytokine receptor systems
The cytokine receptor superfamily consists of single chain proteins, with considerable similarity in their extracellular domains, also termed cytokine homology domains. Signaling is initiated by hormone induced receptor homo/hetero oligomerization and by immediate transphosphorylation of the associated tyrosine kinases JAK/TYK, followed by activation of STAT or MAP kinase pathways. Little is known, however, how this signal is terminated or attenuated. During the last three years a new family of proteins termed inducible SH2-containing proteins (CIS) and JAK2 binding protein (JAB), or alternatively, suppressors of cytokine signaling (SOCS), that are involved in cytokine signaling attenuation, were cloned and partially characterized. The inhibitory activity of these proteins result from their ability to interact with JAK family members or with tyrosine phosphorylated STAT's or with cytokine receptors, yet the mechanism of their activity is not yet fully understood.
Activation of growth hormome and prolactin receptors by placental lactogens and cognate hormones.
Ruminant and other species' placentas synthesize and secrete unique proteins belonging to the growth hormone/prolactin (GH/PRL) family and are termed placental lactogens (PL's). Although a cognate receptor to PL is yet to be discovered, PL can induce receptor dimerization and subsequent activation of several sources of PRLR and GHR. In this study we are determining the three-dimensional structures of two complexes; a) oGHR:oGH 2:1 complex and b) oGHR:oPL 1:1 complex where the oPL has antagonistic properties (The structural studies are being preformed on the extracellular domains of the relevant receptors). Another intriguing finding that resulted in structural studies is that oPL induces heterodimerization and signaling while complexed with two different receptors, oGHR and oPRLR. This heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling.
Structural studies of OB-R binding epitope
The ob gene product, leptin, is an important circulating signal for regulation of body weight. The biological function of leptin is mediated through its membrane-associated receptor, leptin receptor (OB-R). The extracellular domain consists of 816 amino acid residues and shows significant similarity to the class-1 cytokine receptor superfamily. The extracellular region of OB-R comprises seven domains which some of them are Ig like domain linked to each other. This type of structural topology possesses flexibility that may impose difficulties in the crystallizing and structural studies of OB-R. It has been shown by truncations of the receptor's extracellular domain that the leptin-binding region is localized on a shorter segment. This segment comprising of residues 323-640 that evidently is substantially shorter than the complete extracellular domain maintains similar binding affinity towards leptin thus establishing a minimal leptin binding protein.
Fatty acid binding proteins as a platform for the design of enzymes
This research focuses on the design and total synthesis of catalytic proteins (artificial enzymes) via a "chemical ligation" approach using bovine cellular retinoic acid binding protein (CRABP) as a molecular scaffold. The folded conformation of the synthetic polypeptides is expected to resemble that of the corresponding wild type protein. Natural and non-natural amino acids having desired catalytic and binding properties will be strategically introduced in the primary structure of the synthetic protein to function as catalytic groups organized in a pre-determined three-dimensional shell. In this study, the aldolase activity of the amine groups of synthetically mutated residues within the protein's binding site will be determined.
X-ray structural studies of IgG VH fragments selected by phage display as a tool for peptidomimetics design
In IgG immunoglobulin molecules it is well established that the hypervariable loop H3 (CDR3 from the heavy chain) has the most profound contribution to the interaction with antigens. It has been also shown by three-dimensional structural studies that H3 has the highest conformational and structural variability compared to other CDR loops. Camel immunoglobulins could occur naturally devoid of light chains while still maintaining high affinity antigen-binding properties resulting in the use and utilization of a single VH domain as the smallest recognition module. This VH domain can be used as a template for the generation of phage display libraries, which can be selected, for specific antigen target. Experimental structure determination via X-ray crystallography, computational and synthetic methods tools can be combined towards the design, optimization, and synthesis of peptidomimetics. The structural information of binding and conformation of the heavy chain CDR loops provides a powerful generic tool that in an iterative dialogue among the disciplines of X-ray crystallography, computational chemistry, and synthesis would lead to novel drug discovery.
Structural studies ofhigh affinity systems
The avidin-biotin system is being used extensively as a mediator in an expanding range of biological studies including isolation, localization, cytochemistry, immunoassay and diagnostics, as well as for probing genes. Streptavidin is a bacterial avidin-like protein from Streptomyces avidinii, which also displays exceptionally strong binding properties towards biotin. An intriguing finding suggests pseudo-enzymatic properties in avidin, which "catalyzes" the hydrolysis of selected biotin containing active esters, whereas streptavidin actually protects the same reagents from basic hydrolysis. We are determining the three-dimensional structures of avidin and streptavidin complexed with bitingly analogous which could provide a better understanding of the chemical and structural basis of this phenomena of enzymatic activity of avidin versus hydrolysis protection by streptavidin.
Anti biotin antibody
Murine monoclonal anti-biotin antibodies were produced and show relatively high affinity towards biotin KA~109 M-1. This Ab can serve as a very good substitute of for streptavidin-based immunoassays systems and provides an alternative probe for the avidin-biotin technology. After sequencing the VL and VH chains it was found that two CDR loops in the heavy chain, CDR2 (H2) and CDR3 (H3), share high sequence similarity with the biotin binding motifs in avidin and streptavidin.
Livnah, O., Stura, E.A., Johnson, D.L., Middleton, S.A., Mulcahy, L.S., Wrighton, N.C., Dower, W.J, Jolliffe L.K., and Wilson, I.A. (1996) Functional mimicry of a protein hormone by a peptide agonist: the structure of the EPOR complex to 2.8 Å. Science 273, 464-471.
Ibdah, M., Bar-Ilan, A., Livnah, O., Schloss, J.V., Barak, Z. and Chipman, D.M. (1996) Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry 35, 16282-16291.
Satterthwait, A.C., Cabezas, E., Calvo, J.C., Wu, J.X., Wang, P.L., Chen, S.Q., Kaslow, D.C., Livnah, O., and Stura, E.A. (1996) Constrained synthetic peptide vaccines, In: Peptides: Chemistry, Structure and Biology, Proceedings of the 14th American Peptide Symposium. P.T.P. Kaumaya, R.S. Hodges, eds., pp. 772-773.
Johnson, D.L., Barbone, F.X., Mcmahone, F.J., Tullai, J., Barbone, F.P., Middleton, S.A., , K., Livnah, O., Wrighton, N.C., Dower, W.J., Mulcahy, L.S., Stura, E.A., Wilson, I.A., and Jolliffe, L.K. (1997) A peptide mimetic of erythropoietin: critical residues and description of minimal epitope, In: Peptides: Chemistry, Structure and Biology, Proceedings of the 15th American Peptide Symposium.
He, M.-Y., Gani, M., Livnah, O., Stura, E.A., Coley, J., Beale, D., Wilson, I.A., and Taussig, M.J., (1997) Specificity, sequence and crystallization of an oestrone-3-glucuronide antibody: Similarty in binding anti-steroid antibodies with crystallographically determined structures. Immunology 90, 632-639.
Johnson, D.L., Farrel, F.X., Mcmahon, F.J., Tullai, J., Hoey, K., Wrighton, N.C., Livnah, O., Barbone, F.P., Middleton, S.A., Loughney, D.A., Stura, E.A., Dower, W.J., Mulcahy, L.S., Wilson, I.A., and Jolliffe, L.K. (1998) Identification of a 13 amino acid peptide mimetic of erythropoietin and description of amino acids critical for the mimetic activity of EMP1. Biochemistry 37, 3699-3710.
Livnah, O., Johnson, D.L., Stura, E.A., Farrell, F.X., Barbone, F.P., You, Y., Liu, K.D., Goldsmith, M.A., HE, W., Krause, C., Pestka, S., Jolliffe, L.K., and Wilson, I.A. (1998) An antagonist peptide – EPO receptor complex suggests that receptor dimerization is not sufficient for activation. Nature Struct Biol.5, 993-1004.
Livnah, O., Stura, E.A., Middleton, S.A., Johnson, D.L., Jolliffe, L.K. and Wilson, I.A. (1999) Crystallographic evidence for preformed dimers of erythropoietin receptor prior to ligand activation. Science, 283, 987-990.
Herman, A., Helman, D., Livnah, O., Gertler, A. (1999) Ruminant placental lactogens as antagonists to homologous growth hormone receptors and as agonists to human or rabbit growth hormone receptors. J. Biol Chem. 274(12):7631-9.
Middleton, S.A., Barbone, F.P., Johnson, D.L.,Thurmond, R.L., You, Y., McMahon, F.J., Zin, R., Livnah, O., Tulai, J., Farrell, F.X., Wilson, I.A., Goldsmith, M.A., and Jolliffe, L. K. (1999) Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide. J Biol Chem. 274, 14163-9.
Wilson, I.A., Livnah, O., Stura, E.A., Johnson, D.L. and Jolliffe, L.K. "Small Molecule Mimetics of Erythropoietin", U.S. Patent #5835382
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