Structural Biology and Cell Signalling

Keywords: signal transduction, cell adhesion, gene transcription, cytoskeleton assembly, calcium signalling, phospho-inositol signalling, mTOR signalling, NMR, X-ray crystallography, FRET imaging, bioinformatics.

Creating and maintaining tissue organization requires strict control of three processes governed by cellular signalling, namely: cell division, cell differentiation and growth. Cells which escape .“normal” controls and proceed along a path of uncontrolled growth and migration, often lead to cancer. Our laboratory is interested in elucidating the molecular and structural mechanisms underlying signal transduction processes in living systems and the atomic/molecular cause of signalling malfunction caused by genetic disorders and/or environmental stress In order to achieve our goal, we are currently investigating the structure-function relationships of key signalling proteins by various biochemical and biophysical methods, including nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and fluorescence resonance energy transfer (FRET) imaging microscopy. Such research tools enable us to visualize structural details of key protein players and dynamics and the kinetics of their interaction critical to their signalling function. Presently our research focuses on proteins involved in cancer signalling, calcium signalling, cell adhesion, cytoskeleton dynamics and transcriptional regulation. We believe that such molecular studies will ultimately contribute to our understanding of human disease including cancer, neurological disorders, heart disorders and infectious diseases. Details of specific studies are summarized below:

Calcium signalling (supported by CIHR and HSF):

Signalling systems transform external signals such as hormones, growth factors and neurotransmitters into intracellular secondary messengers. By turning specific pathways on or off, these secondary messengers, including Ca2+ and inositol 1,4,5-trisphosphate (IP3), serve a number of distinct cellular functions. The concentration of Ca2+ in the cell is highly regulated by various Ca2+ transporters; therefore, to understand Ca2+ signalling, we need to investigate these transporters and the precise mechanism of Ca2+ homeostasis in the cell. Currently, a major focus is on the store-operated Ca2+ entry (SOCE) mechanism, which plays a pivotal role in T cell activation, as well as many other cellular processes. Recent studies in the field identified STIM1 (stromal interaction molecule-1) and Orai1 (also called CRACM1) as key components of the Ca2+ -release-activated Ca2+ (CRAC) channels which mediate SOCE in T-cells. Upon the Ca2+ depletion of the endoplasmic reticulum (ER) by the IP3 receptor, STIM1 senses the low concentration of Ca2+ in the ER and sends a signal to the plasma membrane pore forming channel Orai1, leading to the Ca2+ influx. A point mutation of Orai1 (R91W) impairs the CRAC activity and is linked to the severe combined immune deficiency syndrome (SCID). We employ structural methodologies to elucidate the molecular basis of this Ca2+ signalling process in order to understand (i) how STIM1 functions as an ER Ca2+ sensor, (ii) how STIM1 communicates with the plasma-membrane Orai1 channel to activate the CRAC function, and (iii) how the SCID mutation may lead to disruption of the normal T cell function.

In response to Ca2+ increase in the cell controlled by the aforementioned mechanism for instance, calmodulin (CaM), a central member of the Ca2+ signalling network, binds Ca2+ and undergoes a conformational change. Normally it is the Ca2+ bound form of the protein that activate (or deactivate) targets in the cell. By turning specific pathways on or off through interaction with numerous downstream proteins, including serine/threonine protein kinases (CaM kinases I, II, IV, CaM kinase kinase, myosin light chain kinases), protein phosphatases (calcineurin and RDGC), nitric oxide synthetase, NMDA receptor, IP3 receptors and Ca2+ channels, CaM essentially functions as a ubiquitous intracellular sensor of Ca2+ . We are now investigating the mechanism by which this ubiquitous Ca2+ sensor protein recognizes and regulates the biological activity of such diverse protein targets.

In our bioinformatics research efforts, we have established the “Cellular Calcium Information Server” (http://calcium.uhnres.utoronto.ca), which currently features databases for calmodulin target proteins, the cadherin superfamily and the EF-hand protein superfamily.

Cell adhesion and cytoskeletal dynamics (supported by NCIC):

The ability of cells to adhere to one another is fundamental to normal cell development and morphogenesis of multicellular organisms. Cancer pathology causes the loss of such cellular adhesion function, permitting the spread of cancer cells from the primary cancer site to other locations in the body (the process referred to as metastasis). Cell adhesion is coupled with intracellular signalling and cytoskeletal assembly. Such cellular processes together control the shape and motility of cells in a dynamic manner. In order to understand the normal cell adhesion process and the associated defects which may occur in cancer cells, we are investigating one of the essential cell-cell adhesion molecules, cadherin, and its binding partners, catenins. We plan (1) to determine the structural basis of the cadherin-mediated cell-cell interaction as well as the role of Ca2+ in this process and, (2) to determine the mechanisms by which the cadherin function is regulated through interaction with intracellular proteins and cytoskeletal components. Presently, we are also studying the microtubule associated protein EB1 and its binding partners in order to elucidate mechanisms underlying microtubule assembly during cell cycle progression. Our ultimate goal in these studies is to fully understand the mechanisms underlying normal cell adhesion and cancer metastasis with the objective of providing fundamental information necessary to support the development of therapeutics intended to eliminate the spread of cancer.

Transcriptional regulation (supported by CIHR):

Cancer, in common with many other diseases, has a genetic component. Genetic information encoded in cellular DNA is transcribed and translated by complex molecular mechanisms. Cancer viruses directly target transcriptional machinery of host cells. In human and other eukaryotes, gene transcription is controlled by an array of general transcription factors (GTFs) and RNA polymerases. TFIIB and TFIID, two transcriptions factors, are highly ubiquitous from yeast to humans and play crucial roles in transcription initiation and activation processes. Presently, we are interested in how such TGFs mediate transcription and participate in the activation of various genes. We are also interested in the internal regulatory mechanism of TFIID by TATA-box binding protein (TBP), TBP-associated factors (TAFs) and the TFIIB-mediated transcriptional activation process. The adenoviral oncoprotein, E1A, and the herpes simplex virus protein, VP16, both directly interact with TBP or TFIIB. This interaction regulates various gene transcription activities. Currently, we are investigating the structure and kinetics of the interaction of such viral transcription regulators with the GTFs. Such information will, we believe, permit us to fully understand the molecular actions of foreign gene products in the host cells, leading to development of methods by which we can prevent unfavourable invasion of cells. Our most recent research efforts involve transcriptional activators and co-activators that are implicated in leukemia development (i.e. AML1-ETO).

Structural Biology of Cancer Signalling (CFI & CRS):

We are part of the newly funded research program on Genomic Instability and Cancer Survival at the Advanced Medical Discovery Institute within OCI. This program will enhance our research activities ultimately contributing to the effective treatment of cancer. To this end, we have initiated several structural projects on cancer targets in collaboration with other OCI scientists. In 2006, a state-of-the-art actively-shielded 800-MHz NMR instrument equipped with a high-sensitivity triple-resonance cryogenic probe was installed. More recently a 600 MHz NMR instrument equipped with high-throughput screening capability has also been installed at the OCI. These instruments are crucial for (a) the investigation of larger proteins which are soluble only at low protein concentrations (many cancer-related proteins fall into this category of targets) and (b) discovery of new chemical inhibitors or biologically relevant interacting molecules of a protein of interest. Linking detailed structural analysis with molecular and cellular functional analyses of cancer proteins, will potentially permit us to make a significant contribution in the fight against cancer. Our current research focus involves structural and functional analysis of Rheb, a small G-protein which acts as a regulator of mTOR.

In 2009, in collaboration with Dr. Vuk Stambolic’s laboratory at the OCI, we have developed a new NMR methodology for assaying the GTPase enzymatic reaction of Rheb using a naturally occurring ligand, GTP (Marshall et al. Sci. Sig. 2009). Rheb belongs to a large family of small G proteins which includes Ras, Rho, Rab, Ran, and Arf. These proteins are active only when it is bound to GTP but inactive when bound to GDP. This molecular switch is controlled by the GTPase cycle which involves a GTP hydrolysis reaction and a GDP-to-GTP exchange reaction. These reactions are catalyzed by two types of enzymes called GAP (GTPase activating protein) and GEF (guanine nucleotide exchange factor). With the new NMR tool in hands, we now can assay both GAP- and GEF-mediated reactions of GTPases using natural ligands GTP and GDP. These studies are currently being expanded towards Ras, RhoA, and other small G proteins.

Stress response and defence mechanisms in bacteria (supported by CIHR):

The signal transduction and gene expression systems are intimately coupled via sensing mechanisms which detect environmental changes or exposure to “environmental stress”. Bacteria provide a simple yet elegant case study of biological strategies involved in sensing and response to stress. The His-Asp phosphorelay system, or the two-component system, involving the sensor histidine kinase and the response regulator are essential to bacterial survival. This signal transduction system is essential to many prokaryotic organisms, but is not found in eukaryotes. For this reason, the His-Asp phosphorelay system is an excellent model which can be used to support antibiotic development. We are currently investigating the structure and mechanism of the EnvZ-OmpR two-component system which serves as an osmosensor in E. coli. Most recently, we have initiated a project on a newly identified class of proteins called ‘addiction modules’ which consist of toxin and anti-toxin operon gene pairs. MazF/MazE and RelE/RelB are well-known examples of addiction module proteins and we are currently investigating their function and molecular mechanisms of their toxicity. These studies could lead to a novel therapeutic approach to treatment of infectious disease - a frequent cause of death in cancer patients due to their compromised immune system.

Summary:
Three-dimensional structural information of proteins can provide useful clues which can help in the design of new therapeutic compounds intended to specifically inhibit certain protein activities. An example of such effort, is the development of new antibiotics targeted for bacteria-specific signal transduction systems involving histidine kinases. In the coming years, we hope to determine 3D structures of more protein-protein complexes by NMR or X-ray crystallography to visualize, via FRET imaging microscopy, specific protein-protein interactions in various cells, including tumour cells. Such research endeavours will lead to a better understanding of cell signalling mechanisms critical for normal human physiology and may also, we hope, result in enhanced therapeutic strategies for cancer and other diseases.

For further information, visit our laboratory website at: http://nmr.uhnres.utoronto.ca/ikura

Graduate Students:

• Fernando Amador
• Jenny Chan
• Bryan Kim (Co-supervisor)
• Mohammad Mazhab Jafari
• Feng Wang
• Le Zheng

Selected Publications:

Link to Pubmed Publications

Marshall CB, Ho J, Buerger C, Plevin MJ, Li GY, Li Z, Ikura M, Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR. (2009) Science Signal. 2(55):ra3, 1-11.

Stathopulos PB, Zheng L, Li GY, Plevin MJ, Ikura M. Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry. (2008) Cell, 135(1): 110-22.

Hayashi I, Plevin MJ, Ikura M. CLIP170 autoinhibition mimics intermolecular interactions with p150Glued or EB1 (2007) Nat Struct Mol Biol.14(10):980-1.

Plevin, M.J., Zhang J., Guo C., Roeder R.G., Ikura M. The acute myeloid leukemia fusion protein AML1-ETO targets E proteins via a paired amphipathic helix-like TBP-associated factor homology domain (2006) Proc Natl Acad Sci USA. 103(27):10242-7.

Ikura, M. Ames, J.B. Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: two ways to promote multifunctionality (2006) Proc Natl Acad Sci USA.103(5):1159-64.

Hayashi, I., Wilde A., Mal T. K., Ikura M. Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex (2005) Mol. Cell, Aug. 19: (19)4: 449-460.

Bosanac I, Yamazaki H, Matsu-Ura T, Michikawa T, Mikoshiba K, Ikura M. Crystal structure of the ligand binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor. (2005) Mol Cell, Jan. 21;17(2): 193-203.

Plevin MJ, Mills MM, Ikura M. The LxxLL motif: a multifunctional binding sequence in transcriptional regulation. (2005) Trends Biochem Sci. 2005 Feb;30(2): 66-9.

Mizuno H, Mal TK, Tong KI, Ando R, Furuta T, Ikura M, Miyawaki A. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. (2003) Mol Cell. Oct;12(4):1051-8.

Bosanac I, Alattia JR, Mal TK, Chan J, Talarico S, Tong FK, Tong KI, Yoshikawa F, Furuichi T, Iwai M, Michikawa T, Mikoshiba K, Ikura M. (2002) Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature 420 , 696-700.

Hoeflich KP, Ikura M. (2002) Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108: 739-742.

Truong K, Sawano A, Mizuno H, Hama H, Tong KI, Mal TK, Miyawaki A, Ikura M. (2001) FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule. Nat. Struct. Biol. 8, 1069-1073.

Tepass U, Truong K, Godt D, Ikura M, Peifer M. (2000) Cadherins in embryonic and neural morphogenesis. Nat. Rev. Mol. Cell. Biol. 1, 91-100.

Osawa M, Tokumitsu H, Swindells MB, Kurihara H, Orita M, Shibanuma T, Furuya T, Ikura M. (1999) A novel target recognition revealed by calmodulin in complex with Ca2+ -calmodulin-dependent kinase kinase. Nat. Struct. Biol. 6, 819-824.

Liu, D., Ishima, R., Tong, K. I., Bagby, S., Kokubo, T., Muhandiram, D. R., Kay, L. E., Nakatani, Y., and Ikura, M. (1998) Solution Structure of a TBP-TAFII230 Complex: Protein Mimicry of the Minor Groove Surface of the TATA Box Unwound by TBP. Cell 94, 573-583.

Tanaka, T., Saha, S. K., Tomomori, C., Ishima, R., Liu, D., Tong, K. I., Park, H., Dutta, R., Qin, L., Swindells, M. B., Yamazaki, T., Ono, A. M., Kainosho, M., Inouye, M., and Ikura, M. (1998) NMR Structure of the Histidine Kinase Domain of the E. coli Osmosensor EnvZ. Nature 396, 88-92.

Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M. and Tsien, R. (1997) Cameleons: Fluorescent Indicators for Ca2+ Based on Green Fluorescent Proteins and Calmodulin. Nature 388, 882-887.

Nagar B, Overduin M, Ikura M, Rini JM. (1996) Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 380, 360-364.

Bagby, S., Kim, S., Maldonado, E., Tong, K. I., Reinberg, D., and Ikura, M. (1995) The Solution Structure of the Carboxy-terminal Core Domain of Human TFIIB: Similarity to Cyclin A and Interaction with the TATA Binding Protein, Cell 82, 857-867.

Tanaka, T., Ames, J.B., Harvey, T.S., Stryer, L. and Ikura, M. (1995) Sequestration of the Membrane-targeting Myristoyl Group of Recoverin in the Calcium-free State, Nature 376, 141-448.

Overduin M, Harvey TS, Bagby S, Tong KI, Yau P, Takeichi M, Ikura M. (1995) Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science 267, 386-389.