Research Description

Structural Studies of Disordered Proteins

Disordered Proteins

Structural studies of intrinsically disordered proteins and regions, which play critical biological roles, lag far behind studies of folded proteins. A major focus of my work has been to bridge this gap. We developed the ENSEMBLE program to generate ensembles of conformers representing a disordered state [Marsh & Forman-Kay, Ensemble modeling of protein disordered states: Experimental restraint contributions and validation, Proteins (2012); Krzeminski et al, Characterization of disordered proteins with ENSEMBLE. Bioinformatics (2013)]. Our contributions to the field of disordered states are described in a recent review [Forman-Kay & Mittag, From sequence and forces to structure, function and evolution of intrinsically disordered proteins, 20th anniversary issue of Structure (2013)].

Dynamic Complexes

Disordered proteins often bind targets in highly dynamic complexes. We demonstrated that the disordered Sic1 cyclin dependent kinase inhibitor binds to the Cdc4 component of an SCF ubiquitin ligase complex in a dynamic complex with multiple phosphorylation sites of Sic1 exchanging on and off of the Cdc4 binding site [Mittag et al, Dynamic equilibrium engagement of a polyvalent ligand with a single site receptor, PNAS (2008); Tang et al, Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase, PNAS (2012)] and have calculated models of the free Sic1 and its dynamic complex with Cdc4, shedding insight into the ultrasensitive ubiquitination of Sic1 within the SCF ligase [Mittag et al, Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase, Structure (2010)]. We characterized Abp SH3 domain complexes involved in actin organization and correlated the degree of engagement within the dynamic complexes with functional data [Stollar et al. Structural, functional and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p. J Biol Chem (2009); Stollar et al, Differential dynamic engagement within 24 SH3 domain:peptide complexes revealed by co-linear chemical shift perturbation analysis, PLoS One (2012)]. We demonstrated a dynamic interaction of 4E-BP2 that is key for translational regulation [Lukhele et al, Interaction of the eukaryotic initiation factor 4E with 4E-BP2 at a dynamic bipartite interface, Structure (2103)].

Importantly, we showed that phosphorylation induces folding of 40 residues of 4E-BP2 to a 4E-binding-incompatible state [Bah et al, Phosphorylation-Induced Folding in an Intrinsically Disordered Protein as a Regulatory Switch, Nature (2014)], the first time significant folding of an IDP due to post-translational modification has been reported. We have also characterized the phase separation of the disordered region of Ddx4, helping to develop this new field that links biophysics and cell biology [Nott et al, Phase transition of a disordered Nuage protein generates environmentally responsive membraneless organelles, Mol Cell (2014)].

Our studies of dynamic complexes have been highlighted in reviews [Mittag et al, Protein dynamics and conformational disorder in molecular recognition, J Mol Recognit (2010); Marsh et al, Probing the diverse landscape of protein flexibility and binding, Curr Opin Struct Biol (2012)].

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Our studies of the dynamic interactions of the CFTR disordered R region led to a model for phospho-regulation of its activity and processing [Bozoky et al, Regulatory R region of the CFTR choloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions, PNAS (2013)]. Studies of the NBD1 with its dynamic regulatory elements provide insights into the molecular basis of CF [Kanelis et al, NMR evidence for differential phosphorylation-dependent interactions of wild type and ΔF508 CFTR, EMBO J (2010); Dawson et al, Allosteric coupling between the ICL4 and regulatory sites of the NBD1 of CFTR, PLoS One (2013)] and the mechanism of action of CF modulator compounds [Hudson et al, Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator, J Biol Chem (2012)]. Our contributions to understanding CFTR structure and dynamics are highlighted in reviews [Chong et al, Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability, Cold Spring Harb Perspect Med (2013); Bozoky et al, Structural changes of CFTR R region upon phosphorylation: a plastic platform for intramolecular and intermolecular interactions, FEBS J (2013)].

Publications

View all publications on PubMed

Conformational Changes Relevant to Channel Activity and Folding within the NBD1 of CFTR
R.P. Hudson, P.A. Chong, I. Protassevitch, R. Vernon, H. Bihler, M. Mense, E. Noy, H. Senderowitz, P.J. Thomas, C. Brouillette and J.D. Forman-Kay
Journal of Biological Chemistry, 287(34):28480-28494 (2012)

Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions
Z. Bozoky, M. Krzeminski, R. Muhandiram, J. Birtley, A. Al-Zahrani, P.J. Thomas, R.A. Frizzell, R.C. Ford and J.D. Forman-Kay
Proc Natl Acad Sci USA, 110(47):E4427-36 (2013)

Phase transition of a disordered Nuage protein generates environmentally responsive membraneless organelles
T.J. Nott, E. Petsalakis, P. Farber, D. Jervis, E. Fussner, A. Plochoweitz, T. Craggs, D. Bazett-Jones, T. Pawson, J.D. Forman-Kay*, A.J. Baldwin
Mol Cell, 57(5):936–947 (2015)

Folding of an Intrinsically Disordered Protein by Phosphorylation as a Regulatory Switch
A. Bah, R. Vernon, Z. Siddiqui, M. Krzeminski, R. Muhandiram, C. Zhao, N. Sonenberg, L.E. Kay, J.D. Forman-Kay
Nature, 519(7541):106–9 (2015)

Deletion of Phenylalanine-508 in the First Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization
P.A. Chong, P.J. Farber, R. Vernon, R.P. Hudson, A.K. Mittermaier and J.D. Forman-Kay
Journal of Biological Chemistry, 290(38): 22862-22878 (2015)

An Allosteric Conduit Facilitates Dynamic Multisite Substrate Recognition by the SCFCdc4 Ubiquitin Ligase
V. Csizmok, S. Orlicky, J. Cheng, J. Song, A. Bah, N. Delgoshaie, H. Lin, T. Mittag, F. Sicheri, H.S. Chan, M. Tyers, J.D. Forman-Kay
Nature Communications, 8, 13943 DOI: 10.1038/ncomms13943 (2017)

Synergy of cAMP and calcium signaling pathways in CFTR regulation
Z. Bozoky, S. Ahmadi, T. Milman, M. Di-Paola, S. Pasyk, R. Pekhletski, J.P. Keller, C.E. Bear, J.D. Forman-Kay
Proc Natl Acad Sci USA, 114(11):E2086-E2095 (2017)

An Evolutionary Switch in ND2 enables Src kinase regulation of NMDA receptors
D.P. Scanlon, A. Bah, M. Krzeminski, W. Zhang, H.L. Leduc-Pessah, Y.N. Dong, J.D. Forman-Kay, M.W. Salter
Nature Communications, 8, 15220 DOI: 10.1038/ncomms15220 (2017)

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations
R.M. Vernon, P.A. Chong, H. Lin, Z. Yang, Q. Zhou, A.A. Aleksandrov, J.E. Dawson, J.R. Riordan, C.G. Brouillette, P.H. Thibodeau, J.D. Forman-Kay
Journal of Biological Chemistry, DOI: 10.1074/jbc.M116.772335 [Epub ahead of print] (2017)

Structural and hydrodynamic properties of an intrinsically disordered region of a germ-cell specific protein upon phase separation
J. Brady, P.J. Farber, A. Sekhar, Y.-H. Lin, R. Huang, A. Bah, T.J. Nott, H.S. Chan, A.J. Baldwin, J.D. Forman-Kay, L.E. Kay
PNAS, in press (2017)