|From left to right : Jing Liu, Louis LeCour, Tomas Vanagunas, and David Worthylake|
Metastatic disease is the leading cause of death for those initially diagnosed with cancer. A necessary step in metastasis is the loss of cell-cell and cell-matrix interactions prior to scattering and invasion of distant tissues. Both the Rac-specific guanine nucleotide exchange factor Tiam1 (177kD) and a novel Rac1/Cdc42 effector, IQGAP1 (190kD) have been implicated in affecting the integrity of cell-cell junctions and the processes of cell migration and invasion. Each of these proteins contains multiple distinct domains and several binding partners have been identified for each protein. Notably, IQGAP1 directly competes with α-catenin for binding to β-catenin, and through removal of α-catenin from cadherin-based cell-cell junctions, IQGAP1 severs the connection to the cytoskeleton thereby loosening intercellular junctions. Less is known about how Tiam1 destabilizes cell-cell junctions, but its activity in this capacity also appears to impinge upon β-catenin function. Dr. Worthylake's research utilizes a structural approach to focus on the molecular mechanisms by which IQGAP1 and Tiam1 destabilize cell-cell junctions.
This is being accomplished using a "divide and conquer" strategy in which protein fragments encompassing one to several complete domains from Tiam1 and IQGAP1 are being expressed and purified from bacteria. Recombinant proteins will be used to identify novel binding partners in cells and will be tested for their ability to crystallize alone and in complex binding partners. X-ray crystallography will then be used to determine the three-dimensional structures of crystallized proteins. As a complement to the structural studies, site directed mutagenesis will be employed to identify residues on IQGAP1, Tiam1 and their activators/effectors that are required for binding and biological activity.
The Figure below shows the Rac1/Tiam1complex that was recently determined by Drs. Worthylake, Rossman and Sondek (Nature408: 682 - 688)
Surface representation of the Tiam1/Rac1 complex. a, Coil representation of the complex b, A GRASP surface representation of the Tiam1/Rac1 complex in the same orientation as in a, illustrating the accessibility of the nucleotide-binding cleft of Rac1 while bound to Tiam1. The GTP analogue (magenta) and Mg2+ (blue) were positioned by aligning the structure of Rac1/GMPPNP onto Rac1 of the complex using a least squares superposition of alpha-carbons from residues 6-24, 52-56, 76-118 and 137-175 (r.m.s. deviations, 0.5 Å). The visibility of the base, ribose, all three phosphates and part of the Mg 2+ illustrate the extensive solvent exposure of the active site.
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