John Sondek, PhD

• Sondek Lab Website
• Publication List

RESEARCH INTERESTS:

Dbl-Family Proteins and Rho GTPases

Rho GTPases act as binary switches to coordinate cytoskeletal alterations with transcriptional events to control numerous cellular processes including: chemotaxis, proliferation, differentiation, phagocytosis, and apoptosis. Rho proteins are inactive when bound to GDP while the exchange of GDP for GTP activates Rho proteins by enhancing affinities for various downstream effectors. Due to the central function of bound guanine nucleotides in controlling downstream signaling, nucleotide-bound states of Rho proteins are regulated tightly. Guanine nucleotide dissociation inhibitors, or GDIs, bind G proteins to prevent spurious activation; GTPase activating proteins, or GTPases, enhance intrinsic rates of GTPase activity within G proteins, and guanine nucleotide exchange factors, or GEFs, are required to catalyze the productive exchange of bound GDP for GTP. Given the central role of Rho GTPase in numerous cellular processes, it is not surprising that aberrant regulation leading to constitutive activation of Rho GTPase is associated with numerous pathologies, including cancer. For example, overly active GEFs specific for Rho GTPase are routinely isolated as oncogenes. In order to understand the molecular details of Rho GTPase activation, we are characterizing several GEFs specific for members of the Rho GTPase family.

The sixteen distinct Rho GTPase, including the highly studied RhoA, Cdc42, and Rac1, comprise one branch of the very large family of Ras-like G proteins. Specific Rho GTPase are activated by exchange through interaction with a large family of GEFs typified by its founding member, Dbl.  There are over 50 unique Dbl-family GEFs possessing a spectrum of specificities toward Rho GTPases. For example Tiam1 catalyzes nucleotide exchange only on Rac1, while Dbs actively exchanges on Cdc42 and RhoA, and Vav is active on all three Rho GTPases. We are intensely interested in understanding the molecular details of the exchange process and how this information relates to biological functions. Such information will allow us to understand: a) how to manipulate guanine nucleotide exchange reaction for therapeutic effects, and b) predict functional pairings of GEFs and G proteins and resultant physiological effects.

Towards these goals, we have solved atomic-resolution crystal structures of several GEF/Rho protein pairs: Tiam1/Rac1, Dbs/Cdc42, intersectin/Cdc42, and Dbs/RhoA and are using this information to guide further studies to understand and manipulate the exchange process in vivo as well as in vitro (e.g. JBC, 276, 27145-51 (2001)). In the near future, we will be using our acquired knowledge and expertise to screen libraries of low-molecular-weight compounds for inhibitors of guanine nucleotide exchange catalyzed by Dbl-family members using high-throughput fluorescence techniques. These inhibitors will then be useful as reagents to probe GEF activities in vivo as well as provide initial leads for potential drug development designed to intervene at the level of Rho GTPase activation.

Heterotrimeric G Protein Effectors

Heterotrimeric G proteins constitute a unique class of GTP-binding proteins distinct from the low-molecular weight GTPase such as Rho and Ras. The extremely large family of G protein-coupled receptors (GPCRs) sense a diverse array of extracellular stimuli and couple this information to conformation changes within the receptors that are transduced across cellular membranes and sensed directly by heterotrimeric G proteins. In essence, the GPCRs acts as GEFs for heterotrimeric G proteins, much the same way Dbl-family proteins function on Rho GTPase as described above. Upon interaction with activated GPCRs, heterotrimeric G proteins exchange GDP bound to the a-subunits for GTP and the heterotrimers dissociate into Ga-GTP subunit and Gbg dimers. Both portions of the dissociated heterotrimers are active in downstream signaling by direct interaction with effectors. b isozymes, G-protein coupled receptor kinases (GRKs), and Tek-family kinases.  Initial success in this area has led to the first atomic resolution structure of the unique C-terminal domain of PLC-b. This domain is the major determinant for binding activated Gaq family members leading to enhanced PLC-b activity. Furthermore, the C-terminal domain of PLC-b also acts as a GTPase activating protein and stimulates the intrinsic hydrolytic rate of Gaq subunits. In other words, PLC-b is not only a G protein effector, but also a GAP and these seemingly mutually antagonistic actions function to enhance the signal-to-noise ratio of the transduction cascade. In summation, by studying the structural bases for activation of effectors by G proteins, we are increasing our understanding of both: a) mechanisms used by G proteins to stimulate effectors, and b) mechanisms used by effectors to reciprocally regulate G proteins.

In contrast to the wealth of existing structural data describing heterotrimeric G protein subunits and their interaction with each other, there is relatively little information regarding the interaction of G proteins with downstream effectors or GPCRs. In order to address this lack of understanding, we have been focusing on structural studies of classical effectors for G proteins including phospholipase C-.

Engineering b-Propeller Scaffolds

Often, the majority of biological activity associated with protein complexes is limited to a small portions of the interacting proteins.  Consequently, it is often possible to graft epitopes into heterologous proteins while simultaneously transferring functions associated with the grafted epitopes. This ability has widespread potential spanning the gamut from understanding the structural determinants dictating particular protein structures to developing small peptitomimetic therapeutics designed to alter protein interactions. Unfortunately, current knowledge has produced few rules for successfully manipulating protein architectures for experimental research and development applications. Furthermore, grafted epitopes are often presented in small protein scaffolds designed for thermodynamic stability and associated experimental tractability resulting in greatly limited ability to present multiple protein sequences associated with diverse binding or catalytic activities. Consequently, we are interested in understanding how to manipulate protein structures to present multiple peptide epitopes with regiospecificity. This ability will allow researchers to coordinate spatially several binding events of substrates and products leading to enhanced effective concentrations of reactants and controlled enzymatic reactions more similar to properties of natural scaffolds than is currently available.

We are deriving our experimental inspirations from b-propeller domains, a ubiquitous protein architecture, commonly found coordinating multifarious protein components to control diverse signaling cascades. In particular we are focusing on b-propellers composed almost entirely of WD-repeats, a primary amino acid sequence repeat that is highly conserved, normally about 40 amino acids in length, and often terminated with a signature tryptophan-aspartate (WD) sequence pair. We are interested in understanding the structural determinants that instruct the proper folding and stability of b-propellers composed of WD-repeats so that we can then use this repeating sequence to build large protein scaffolds with independently addressable sites for the binding of distinct protein components.

In sequenced genomes, domains composed of WD-repeats are typically highly abundant and it is somewhat surprising that there is such a paucity of useful studies designed to understand the physico-chemical properties of this domain architecture. Consequently, we are focusing on the few WD-repeat-containing domains that are relatively well characterized and experimentally tractable, including the b-subunit of heterotrimeric G proteins; receptors for activated C kinase, or Racks; and the yeast protein, Sec13p, required for proper secretory responses.

We are taking several approaches to delimit the structural requirements necessary for the intradomain interactions between repetitive arrays of WD-repeats folding into b-propeller structures. As one approach, we are randomly mutating chimeric b-propellers and identifying stably folded proteins.

As a second approach, we are replacing select WD-repeats within Rack1 and Sec13p with libraries of synthetic oligonucleotides encoding degenerate sequences constrained by the amino acid distributions found in naturally occurring WD-repeats. Similar to above, soluble chimeric versions will be sequenced to determine sequence combinations allowing stable folding of WD-repeat-containing domains.

To facilitate oligonucleotide production, we are using trinucleotide phosphoramidites synthons as opposed to standard mononucleotide chemistries. Since we are solely concerned with the amino acid distribution of the encoded oligonucleotides, trinucleotide synthons corresponding to codons facilitate production of oligonucleotide pools with essentially no bias of amino acid distributions typically encountered when using mononucleotide phosphoramidites as building- blocks.

RECENT PUBLICATIONS:

Cheever ML, Snyder JT, Gershburg S, Siderovski DP, Harden TK, Sondek J. Crystal structure of the multifunctional Gbeta5-RGS9 complex. Nat Struct Mol Biol. 2008 Jan 20. Epub 2008

Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nat Struct Mol Biol. 2007 Sep;14(9):814-23. Epub 2007

Rojas RJ, Yohe ME, Gershburg S, Kawano T, Kozasa T, Sondek J. Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain. J Biol Chem. 2007 Oct 5;282(40):29201-10. Epub 2007

Desveaux D, Singer AU, Wu AJ, McNulty BC, Musselwhite L, Nimchuk Z, Sondek J, Dangl JL. Type III effector activation via nucleotide binding, phosphorylation, and host target interaction. PLoS Pathog. 2007 Mar;3(3):e48

Chhatriwala MK, Betts L, Worthylake DK, Sondek J. The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation. Mol Biol. 2007 May 18;368(5):1307-20. Epub 2007

Yohe ME, Rossman KL, Gardner OS, Karnoub AE, Snyder JT, Gershburg S, Graves LM, Der CJ, Sondek J. Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif. J Biol Chem. 2007 May 4;282(18):13813-23. Epub 2007

Bencharit S, Cui CB, Siddiqui A, Howard-Williams EL, Sondek J, Zuobi-Hasona K, Aukhil I. Structural insights into fibronectin type III domain-mediated signaling. J Mol Biol. 2007 Mar 23;367(2):303-9. Epub 2006

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. 2006 Dec;13(12):1135-40. Epub 2006

Baumeister MA, Rossman KL, Sondek J, Lemmon MA. The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity. Biochem J. 2006 Dec 15;400(3):563-72

Bourdon DM, Wing MR, Edwards EB, Sondek J, Harden TK. Quantification of isozyme-specific activation of phospholipase C-beta2 by Rac GTPases and phospholipase C-epsilon by Rho GTPases in an intact cell assay system. Methods Enzymol. 2006;406:489-99

Snyder JT, Jezyk MR, Gershburg S, Harden TK, Sondek J. Regulation of PLCbeta isoforms by Rac. Methods Enzymol. 2006;406:272-80