Structural and Dynamic Studies of Protein Complexes
We are interested in understanding the basis for sequence-specific protein recognition in the regulation of biological processes, including signal transduction. We have characterized SH2, SH3, PTB and WW domains with their targets and have described "non-canonical" modes of binding from structure of complexes as well as energetic details of the interface based on dynamics and binding experiments. We are now moving towards more detailed studies of the thermodynamic and kinetic effects of disordered target regions in interactions showing cooperativity or avidity. In addition, we are studying a number of multi-domain enzymes, including Eph kinase and the Smurf2 ubiquitin ligase, in order to probe the regulatory effects of intramolecular protein interactions.

Unfolded State of an SH3 domain
In the absence of peptides derived from its binding target, SOS, the N-terminal SH3 domain of the Drosophila drk protein is in equilibrium between folded and unfolded states. We have characterized the structure and dynamics of this unfolded state, which is highly populated under non-denaturing solution conditions, using NMR and other spectroscopic methods and find it to be quite distinct from a theoretical random coil with significant native-like and some non-native residual structure. Insights into protein folding have been derived from our studies. We have exploited this protein as a model system in our development of a computational approach (ENSEMBLE) to facilitate a more quantitative characterization of unfolded and other disordered states and continue to refine this methodology.

Disordered States of Proteins
The primary sequence motifs that are targets for modular binding domain interaction are located within intrinsically disordered protein regions. Other disordered regions contain binding targets that adopt more complex structure upon binding. Disordered regions of proteins have been recognized as important for mediating protein recognition in regulation, with almost half of eukaryotic proteins having significant stretches of disorder. We are characterizing a number of these intrinsically disordered regions of proteins, including those implicated in signal transduction. These include gamma-synuclein (a marker for breast cancer), Sic1 (a cyclin dependent kinase inhibitor), the regulatory (R) region of CFTR, the N-terminal portion of the prion protein and its homologue Shadoo and 4E-BP2 (part of the translation initiation machinery). Knowledge of (i) the polymer chain characteristics of these proteins, (ii) the thermodynamic and structural properties of both free and complexed states and (iii) the range of dynamics remaining in complexes involving disordered proteins is critical for understanding how disordered proteins function in protein recognition.

Domains of CFTR
A third area of interest in my lab is the cytoplasmic regions of CFTR in order to better understand the gating and regulation of function of this chloride channel. In addition to the disordered regulatory R region mentioned above, there are nucleotide binding domains NBD1 and NBD2, with NBD1 being the site of the most common CF mutation, the N-terminal helix and the intracellular loop (ICL) regions. We are currently characterizing the structural, dynamic and interaction properties of the NBD1, NBD2, R region and ICL peptides using NMR methods. We are also probing interactions of these regions with CF therapeutic compounds.

Mittag T, Orlicky S, Choy W-Y, Tang XJ, Sicheri F, Kay LE, Tyers M, Forman-Kay JD. Dynamic equilibrium engagement of a polyvalent ligand with a single site receptor, Proceedings of the National Academy of Sciences 105: 17772-7 (2008).

Bezsonova I, Bruce MC, Wiesner S, Lin H, Rotin D, Forman-Kay JD. Interactions between the three CIN85 SH3 domains and ubiquitin: Implications for CIN85 ubiquitination, Biochemistry 47: 8937-49 (2008).

Marsh JA, Baker JM, Tollinger M, Forman-Kay JD. Calculation of residual dipolar couplings from disordered state ensembles using local alignment. Journal of the American Chemical Society 130:7804-5 (2008).

Wiesner S, Ogunjimi AA, Wang HR, Rotin D, Sicheri F, Wrana JL, Forman-Kay JD. Auto-inhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain, Cell 130:651-62 (2007).

Baker JMR, Hudson RP, Kanelis V, Choy W-Y, Thibodeau PH, Thomas PJ, Forman-Kay JD. CFTR regulatory (R) region interacts with NBD1 predominantly via multiple transient helices, Nature Structural & Molecular Biology 14, 738-45 (2007).

Borg M, Mittag T, Pawson T, Tyers M, Forman-Kay JD, Chan H-S. Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity, Proceedings of the National Academy of Sciences 104, 9650-5 (2007).

Marsh JA, Neale C, Jack FE, Choy W-Y, Lee A, Crowhurst KA, Forman-Kay JD. Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure, Journal of Molecular Biology 367, 1494-510 (2007).

Mittag T, Forman-Kay JD. Atomic-level characterization of disordered protein ensembles, Current Opinion in Structural Biology 17, 3-14 (2007).

Marsh JA, Singh VK, Jia Z, Forman-Kay JD. Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation. Protein Science 15, 2795-804 (2006).

Wiesner S, Wybenga-Groot LE, Warner N, Lin H, Pawson T, Forman-Kay JD, Sicheri F. A Change in Conformational Dynamics Underlies the Activation of Eph Receptor Tyrosine Kinases, EMBO Journal 25, 4686-96 (2006).

Chong PA, Lin H, Wrana JL, Forman-Kay JD. An Expanded WW Domain Recognition Motif Revealed by the Interaction between Smad7 and the E3 Ubiquitin Ligase Smurf2, Journal of Biological Chemistry 281, 17069-75 (2006).

Kanelis V, Bruce MC, Skrynnikov NR, Rotin D, Forman-Kay JD. Structural Determinants for High Affinity Binding in a Nedd4 WW3* Domain-Comm PY Motif Complex, Structure 14, 543-53 (2006).

Bezsonova I, Singer A, Choy WY, Tollinger M, Forman-Kay JD. Structural comparison of the unstable drkN SH3 domain and a stable mutant. Biochemistry 44, 15550-60 (2005).

Tollinger M, Kay LE, Forman-Kay JD. Measuring pK(a) values in protein folding transition state ensembles by NMR spectroscopy. Journal of the American Chemical Society 127, 8904-5 (2005).

Finerty PJ Jr, Mittermaier AK, Muhandiram R, Kay LE, Forman-Kay JD. NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain. Biochemistry 44, 694-703 (2005).