LSUHSC School of Medicine - Biochemistry Laboratory 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 (Nature 408: 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 Mg²⁺ illustrate the extensive solvent exposure of the active site.

Jezyk MR, Snyder JT, Gershberg S, Worthylake DK, Harden TK, Sondek J. Crystal structure of Rac1 bound to its effector phospholipase C-beta2. Nat Struct Mol Biol. 13(12):1135-40. (2006)

Worthylake DK, Rossman KL, Sondek J. Crystal structure of the DH/PH fragment of Dbs without bound GTPase. Structure (Camb)12(6):1078-86 (2004)

Rossman KL, Worthylake DK, Snyder JT, Cheng L, Whitehead IP, Sondek J. Functional analysis of cdc42 residues required for Guanine nucleotide exchange. J Biol Chem. 277(52):50893-8 (2002)

Cheng L, Rossman KL, Mahon GM, Worthylake DK, Korus M, Sondek J, Whitehead IP. RhoGEF specificity mutants implicate RhoA as a target for Dbs transforming activity. Mol Cell Biol. ;22(19):6895-905 (2002)

Snyder JT, Worthylake DK, Rossman KL, Betts L, Pruitt WM, Siderovski DP, Der CJ, Sondek J. Structural basis for the selective activation of Rho GTPases by Dbl exchange factors. Nat Struct Biol. 9(6):468-75 (2002)

Rossman KL, Worthylake DK, Snyder JT, Siderovski DP, Campbell SL, Sondek J. A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange. EMBO J. 21(6):1315-26 (2002)

Karnoub AE, Worthylake DK, Rossman KL, Pruitt WM, Campbell SL, Sondek J, Der CJ. Molecular basis for Rac1 recognition by guanine nucleotide exchange factors. Nat Struct Biol. 8(12):1037-41 (2001)

Worthylake DK, Rossman KL, Sondek J. Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature 408(6813):682-8 (2000)

Worthylake DK, Wang H, Yoo S, Sundquist WI, Hill CP. Structures of the HIV-1 capsid protein dimerization domain at 2.6 A resolution.55:85-92 (1999)

Worthylake DK, Prakash S, Prakash L, Hill CP. Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6 A resolution. J Biol Chem. 273(11):6271-6 (1998)

Gamble TR, Yoo S, Vajdos FF, von Schwedler UK, Worthylake DK, Wang H, McCutcheon JP, Sundquist WI, Hill CP. Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science. 278(5339):849-53 (1997)

Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, Sundquist WI, Hill CP. Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell 87(7):1285-94. (1996)

Massiah MA, Worthylake DK, Christensen AM, Sundquist WI, Hill CP, Summers MF. Comparison of the NMR and X-ray structures of the HIV-1 matrix protein: evidence for conformational changes during viral assembly. Protein Sci.;5(12):2391-8 (1996)

Hill CP, Worthylake DK, Bancroft DP, Christensen AM, Sundquist WI. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly. Proc Natl Acad Sci. U S A.;93(7):3099-104 (1996)

Lehman CW, Clemens M, Worthylake DK, Trautman JK, Carroll D. Homologous and illegitimate recombination in developing Xenopus oocytes and eggs. Mol Cell Biol.;13(11):6897-906 (1993)

Hurley JH, Faber HR, Worthylake DK, Meadow ND, Roseman S, Pettigrew DW, Remington SJ. Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase. Science. 1993;259(5095):673-7.

Worthylake DK, Meadow ND, Roseman S, Liao DI, Herzberg O, Remington SJ. Three-dimensional structure of the Escherichia coli phosphocarrier protein IIIglc. Proc Natl Acad Sci. U S A. ; 88(23):10382-6 (1991)