Meytal Landau - My PhD Thesis

My current website: http://people.mbi.ucla.edu/meytal/

My doctoral work was done between 2002-2007 at the computational lab of Prof. Nir Ben-Tal in the Department of Biochemistry at Tel-Aviv University

And as a Research assistant in Prof. Uri Seligsohn's lab at the Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel.

 

Published projects:
The Regulation Mechanism of the EGF Receptor.

Functional Analysis of the Na+/H+ Transporters.

Identification of Functionally and Structurally Important Residues.

Analyzing the Molecular Effects of Disease-Causing Mutations.

Specific Interactions between Alpha and Beta Subunits of Integrins.

Summery:

The main aim of my doctoral research is to study sequence-structure-function relationships in selected protein families using computational tools. My first interest was in the regulation of the epidermal growth factor receptor (EGFR) family [1]. My computational approach is to combine structural data with the analysis of protein sequences, using methods that take explicitly into account the phylogenetic relations between proteins.

One of my main objectives is to identify functionally and structurally important amino-acid sites, e.g. residues involved in ligand binding, enzymatic activity, and protein-protein interactions, all of which often correspond to evolutionary conserved residues. Such positions can be identified by an evolutionary-conservation analysis projected on the 3-dimensional structure, using the ConSurf web-server (http://consurf.tau.ac.il/). Recently, some methodological improvements were added to ConSurf. I was involved in the integration of these new features into ConSurf, which has led to the publication of ConSurf version 3.0 [2].

In collaboration with experimental laboratories, I also used computational tools to explain the molecular effect of disease-causing mutations [3-11].

 

Published projects:

A Putative Mechanism for Down-Regulation of the Catalytic Activity of the EGF Receptor via Direct Contact between its Kinase and C-Terminal Domains

Meytal Landau, Sarel J. Fleishman and Nir Ben-Tal PubMed

Tyrosine kinase receptors of the EGFR family play a significant role in vital cellular processes and in various cancers. EGFRs are unique among kinases, as the regulatory elements of their kinase domains are constitutively ready for catalysis. Nevertheless, the receptors are not constantly active. This apparent paradox has prompted us to seek mechanisms of regulation that do not involve conformational changes of the kinase domain. Our computational analyses, based on the 3-dimensional structure of EGFRís kinase domain suggest that direct contact between the kinase and a segment from the C-terminal regulatory domains inhibits enzymatic activity. This conclusion is based on a study of the geometric and electrostatic complementarity between the kinases and C-terminal domains within the crystal, and is strengthened by an evolutionary analysis of correlation between amino acid replacements in tyrosine kinase sequences. EGFR activation would then involve temporal dissociation of this stable complex, for example, via ligand-induced contact formation between the extracellular domains leading to the reorientation of the transmembrane and intracellular domains (Figure 1). Thus, the C-terminal domain serves as a down-regulator of the kinase activity in the EGFR family.

Strong reinforcement of this model of regulation is provided by data on human cancer-causing EGFR mutants and viral variants. These EGFR analogues sustained mutations or deletions in the C-terminal segment contacting the kinase domain, which leads to higher autokinase activity and transforming ability. Our model suggests that these mutations destabilize the inactive complex. The model provides an explanation at the molecular level for the effects of cancer-causing EGFR mutations, and suggests a novel therapeutic venue for EGFR-related cancer.

figure1

 

EGFR mechanism

 

Figure 1. Schematic diagram representing the suggested model of EGFR activation.

Two EGFR monomers are colored light purple and yellow. The extracellular domain (residues 1-620, labeled I, II, III, and IV according to its sub-domains) and kinase domain (residues 685-957) are connected via a transmembrane helix (residues 621-642) and a short juxtamembrane segment (not shown). The C-terminal domain, comprising 229 amino acids, whose structure has not been determined, follows the kinase domain. Tyrosine residues (Y) known as the autophosphorylation sites in the C-terminal domain are indicated. In the inactive conformation (left), each of the extracellular domains assumes a compact structure, and the intracellular domains contact via the C-terminal fragments, leading to an inactive and stable form. Activation (right) occurs when ligands (purple ovals) bind to the extracellular domains, leading to the formation of a stable extracellular contact, which is followed by the rotation of the transmembrane helices, and the subsequent destabilization of the contacts between the C-terminal and kinase domains. The kinase can now trans-autophosphorylate the tyrosine residues of its own C-terminal domain, as well as tyrosine residues of its protein substrates.

 

ConSurf 2005: The Projection of Evolutionary Conservation Scores of Residues on Protein Structures

Meytal Landau, Itay Mayrose, Yossi Rosenberg, Fabian Glaser, Eric Martz, Tal Pupko, and Nir Ben-Tal†† PubMed

Key amino acid positions that are important for maintaining the 3-dimensional structure of a protein and/or its function(s), e.g., catalytic activity, binding to ligand, DNA or other proteins, are often under strong evolutionary constraints. Thus, the biological importance of a residue often correlates with its level of evolutionary conservation within the protein family. ConSurf (http://consurf.tau.ac.il/) is a web-based tool that automatically calculates evolutionary conservation scores and maps them on protein structures via a user-friendly interface (See example in Figure 2).

Consurf Kcsa potassium channel

Figure 2. A ConSurf analysis of the Kcsa potassium channel.

The tetrameric channel, which is viewed along the pore from the extracellular end, is presented using a space-filled model. The amino-acids are colored by their conservation-grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved. Amino acid positions, for which the inferred conservation level was assigned with low confidence, are marked with light yellow. The potassium ion at the channel pore is colored green. Conservation scores, which were calculated for one of the channelís subunits, were projected on the homo-tetrameric structure. The run was carried out using PDB code 1bl8 and default ConSurf parameters.

 

Functional Analysis of the Na+/H+ Transporters

Few years ago, a most interesting structure of the Na+/H+ antiporter in E. Coli was determined (Hunte et. al., Nature 435; 1197-1202; 2005) presenting a novel fold. This unique structure encouraged me to examine the Na+/H+ transporter family in order to provide insights on the structural basis of Na+/H+ exchange and its unique regulation by pH. In this project, I will use In-silico methods to guide biochemical experiments to enhance our level of knowledge of processes related to ion-translocation and pH regulation in Na+/H+ transporters. Hopefully, the computational work will be examine by experimental means in collaboration with Prof. Etana Padan (The Hebrew University), who was involved in the determination of the structure, and also with Prof. Rajini Rao (Johns Hopkins University School of Medicine), who is also an expert in this protein family.

 

Analyzing the Molecular Effects of Disease-Causing Mutations

Since the beginning of my doctoral studies, I have been working with the group of Prof. Uri Seligsohn at the coagulation department in Chaim Sheba medical center, Tel Hashomer. My responsibility is to provide the structural and phylogenetic insights to the understanding of regulatory mechanisms and effects of disease-causing mutations in proteins related to coagulation.

I have gained much knowledge in the chemical reaction, structural properties and phylogeny of serine protease coagulation factors as FVII, FIX, FX, and FXI. I was mainly involved in the characterization of mutations leading to bleeding disorders [4, 5, 11], but I was also interested in sites that determine specific properties of serine proteases, as ligand and co-factor binding, regulation and protein-protein interactions.

I have also analyzed mutations in αIIb-β3 integrin, expressed on the surface of blood platelets, leading to Glanzmann Thrombasthenia [7-9]. The work on the very exciting integrin family lead me to conduct research on the evolutionary processes of integrins, in order to identify sites determining specific traits, such as ligand binding and protein-protein interactions.

I have collaborated with Prof. Gideon Rechavi (The Chaim Sheba medical center, Tel-Hashomer and Sackler school of medicine, Tel Aviv University) to explain the molecular effect of human cancer-causing mutations in P53 [3].

In was also involved in the analysis of mutations in the nucleocapsid domain of the Gag polyprotein of HIV- 1, in collaboration with Dr. Eran Bacharach (Department of Cell Research and Immunology, Tel Aviv University) [6].

 

Specific Interactions between Alpha and Beta Subunits of Integrins

Integrins are part of a large family of cell adhesion receptors through which the cell both binds and responds to the extracellular matrix or other cells. Functional integrins consist of two transmembrane glycoprotein subunits, called alpha and beta, which are non-covalently bound. For example, the platelet glycoprotein alpha-IIb beta-3 (integrin αIIb-β3), expressed on nonstimulated platelets, is a functional receptor that mediates selective and irreversible adhesion to immobilized fibrinogen. During my long-term collaboration with Prof. Seligsohn's group at the coagulation department in Tel-Hashomer hospital, I examined mutations in the platelet integrin IIb-IIIa, which cause Glanzmann thrombasthenia, a rare severe bleeding disorder [7-9].

Although integrins share a similar physiological role, they differ in their ligand selectivity, signal transduction and in the affinities between different alpha and beta subunits. It is anticipated that positions that are responsible for these traits are not strictly conserved in evolution, but rather display a unique patterns of substitution. I plan to identify these positions using SpecDet (Fleishman et.al., Mol. Cell 15; 879-888; 2004) and to analyze their functional and structural importance.

 

 

References

1. Landau, M., S.J. Fleishman and N. Ben-Tal, A putative mechanism for down-regulation of the catalytic activity of the EGF receptor via direct contact between its kinase and C-terminal domains. Structure, 2004. 12(12): p. 2265-75. PubMed

2. Landau, M., I. Mayrose, Y. Rosenberg, F. Glaser, E. Martz, T. Pupko and N. Ben-Tal, ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic-Acids-Res, 2005. 33(Web Server issue): p. W299-302. PubMed

3. Ashur-Fabian, O., A. Avivi, L. Trakhtenbrot, K. Adamsky, M. Cohen, G. Kajakaro, A. Joel, N. Amariglio, E. Nevo and G. Rechavi, Evolution of p53 in hypoxia-stressed Spalax mimics human tumor mutation. Proc-Natl-Acad-Sci-U-S-A, 2004. 101(33): p. 12236-41. PubMed

4. Fromovich-Amit, Y., A. Zivelin, N. Rosenberg, H. Tamary, M. Landau and U. Seligsohn, Characterization of mutations causing factor VII deficiency in 61 unrelated Israeli patients. J-Thromb-Haemost, 2004. 2(10): p. 1774-81. PubMed

5. Fromovich-Amit, Y., A. Zivelin, N. Rosenberg, M. Landau, J.P. Rosa and U. Seligsohn, Of four mutations in the factor VII gene in Tunisian patients, one novel mutation (Ser339Phe) in three unrelated families abrogates factor X activation. Blood-Coagul-Fibrinolysis., 2005. 16(5): p. 369-374. PubMed

6. Mark-Danieli, M., N. Laham, M. Kenan-Eichler, A. Castiel, D. Melamed, M. Landau, N.M. Bouvier, M.J. Evans and E. Bacharach, Single point mutations in the zinc finger motifs of the human immunodeficiency virus type 1 nucleocapsid alter RNA binding specificities of the gag protein and enhance packaging and infectivity. J-Virol, 2005. 79(12): p. 7756-67. PubMed

7. Peretz, H., N. Rosenberg, M. Landau, S. Usher, E.J. Nelson, R. Mor-Cohen, D.L. French, B.W. Mitchell, S.C. Nair, M. Chandy, B.S. Coller, A. Srivastava and U. Seligsohn, Molecular diversity of Glanzmann thrombasthenia in southern India: new insights into mRNA splicing and structure-function correlations of alphaIIbbeta3 integrin (ITGA2B, ITGB3). Human Mutation, 2006. In Press. PubMed

8. Rosenberg, N., M. Landau, J. Luboshitz, G. Rechavi and U. Seligsohn, A novel PHE171CYS mutation in integrin aIIB causes Glanzmann Thrombasthenia by abrogating aIIbb3 complex formation. J-Thromb-Haemost, 2004. 2(7): p. 1167-75. PubMed

9. Rosenberg, N., H. Hauschner, H. Peretz, R. Mor-Cohen, M. Landau, B. Shenkman, G. Kenet, B.S. Coller, A.A. Awidi and U. Seligsohn, A 13-bp deletion in alphaIIb gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia. J-Thromb-Haemost, 2005. 3(12): p. 2764-72. PubMed

10. Vysokovsky, A., R. Saxena, M. Landau, A. Zivelin, R. Eskaraev, N. Rosenberg, U. Seligsohn and A. Inbal, Seven novel mutations in the factor XIII A-subunit gene causing hereditary factor XIII deficiency in 10 unrelated families. J-Thromb-Haemost, 2004. 2(10): p. 1790-7. PubMed

11. Zivelin, A., T. Ogawa, S. Bulvik, M. Landau, J.R. Toomey, J. Lane, U. Seligsohn and D. Gailani, Severe factor XI deficiency caused by a Gly555 to Glu mutation (factor XI-Glu555): a cross-reactive material positive variant defective in factor IX activation. J-Thromb-Haemost, 2004. 2(10): p. 1782-9. PubMed