Andrew Lee Lab
Andrew Lee Lab
We study the role of conformational dynamics in protein function, conformational changes, enzyme catalysis, drug binding, and allostery. We use a variety of biophysical and biochemical tools, especially NMR spectroscopy.
Research in the laboratory is centered on understanding the role of structural dynamics in protein function. In past decades, proteins were essentially viewed as static structures. Today, they are widely appreciated to be dynamic ensembles of interconverting structures. Such behavior can be clearly seen in proteins that undergo dramatic shape changes in different functional states. However, the effects of dynamics can also be important when the structural changes are less apparent. The ensemble nature of proteins has far-reaching implications for understanding basic natural protein functions such as ligand binding, enzyme catalysis, and allostery. An understanding of protein dynamics should lead to improvements in protein engineering and rational drug design.
We are particularly interested in how protein dynamics facilitates enzyme catalysis and allosteric communication. To maximize our understanding of these phenomena, we study multiple systems with the idea that different proteins may use different strategies for achieving catalysis and allostery. CheY, a so-called response regulator receiver domain, is the master regulatory switch for reversing the direction of the E. coli flagellum, and it undergoes a classical allosteric conformational change upon phosphorylation of an aspartate side chain. NMR studies from our lab and others indicate these proteins dynamically switch between inactive and active conformations on the microsecond-millisecond timescale, but that the actual switching mechanism involves additional states yet to be characterized (see McDonald et al., Structure, 2012, 20, 1363).
Chorismate mutase (CM) is a metabolic enzyme in the amino acid biosynthesis pathway that exhibits all the hallmarks of allostery. Yet, despite its larger size (60 kDa), it is amenable to detailed study by NMR. CM has a homodimeric structure that has been shown to adopt distinct, classical ‘tense’ (T) and ‘relaxed’ quaternary conformations. CM reactivity is intrinsically positively cooperative, even though the two symmetric active sites are at opposite sites of the dimer, and CM can be both positively and negatively modulated by the allosteric effector ligands tryptophan and tyrosine. Application of NMR spectroscopy to this rich system should enable discovery of structural and dynamic processes that underlie classical allosteric regulation. In particular, we are interested in how the binding event in one protomer can extend to the other protomer active site – via conformational change or other dynamic processes – to stimulate higher activity.
We have been studying the dimeric enzyme thymidylate synthase (or “TS”) for its response to inhibitor/drug binding and its functional allostery. This enzyme is metabolically critical, performing conversion of uridine monophosphate to thymidine monophosphate. Our studies focus on the human and bacterial forms, which show interesting differences in their sequences and substrate binding properties. TS is more complex than many of the enzymes studied to date by NMR, as it has a multi-state reaction coordinate linking substrates and products. This affords an opportunity to track how the dynamics throughout TS change as the enzyme populates different intermediate steps in catalysis. In addition to this interesting reaction mechanism, TS is known to be “half the sites reactive”, meaning that catalytic activity in one subunit imparts non-reactivity to the other subunit. This is essentially a form of high negative cooperativity. Our NMR studies of ecTS (from E. coli) will also focus on this intersubunit negative cooperativity and the structural and dynamic features associated with it. Strikingly, hTS (from human) shows positive cooperativity in binding substrate, while still exhibiting half-site-reactivity. In summary, TS is a fascinating, large enzyme that it interesting from both the perspective of catalysis and allostery.
How do we characterize protein dynamics? Our preferred method is heteronuclear NMR spectroscopy, which is uniquely suited to study both structure and dynamics in proteins and other biological macromolecules. A major advantage of NMR is that spectroscopic probes are distributed uniformly throughout the biomolecule, such as NH or CH atom pairs, providing large amounts of molecular information. To gain information on protein dynamics, NMR spin relaxation is highly sensitive to molecular motion over a range of timescales. We look at the relaxation properties of 15N, 13C, 1H, and 2H spins located throughout the protein scaffold, and interpret these in terms of amplitudes and timescales of individual bond vectors. Slower motions on the microsecond-millisecond timescale can be detected to yield site-specific kinetic, thermodynamic, and structural information on the switching between discrete conformational states. In many cases, these NMR-relaxation dynamics are used to complement other structural data from X-ray crystallography, or thermodynamic and kinetic biophysical measurements using methods such as fluorescence spectroscopy, calorimetry, amide hydrogen exchange, and molecular dynamics simulations.
Wang J, Jain A, McDonald LR, Gambogi C, Lee AL, and Dokholyan NV, Mapping allosteric communications within individual proteins, Nature Communications (2020), 11, 3862.
Bonin JP, Sapienza PJ, Wilkerson E, Goldfarb D, Wang L, Herring L, Chen X, Major MB, and Lee AL, Positive cooperativity in substrate binding by human thymidylate synthase, Biophysical Journal (2019), 117, 1074-1084.
Sapienza PJ, Popov KI, Mowrey DD, Falk BT, Dokholyan NV, and Lee AL, Inter-active site communication mediated by the dimer interface beta-sheet in the half-the-sites enzyme, thymidylate synthase, Biochemistry (2019), 58, 3302-3313.
Lee AL and Sapienza PJ, Thermodynamic and NMR assessment of ligand cooperativity and intersubunit communication in symmetric dimers: application to thymidylate synthase, Frontiers in Molecular Biosciences (2018), 5, article 47. doi: 10.3389/fmolb.2018.00047
Sapienza PJ and Lee AL, Widespread perturbation of function, structure, and dynamics by a conservative single atom substitution in thymidylate synthase, Biochemistry (2016), 55, 5702-5713.
Falk BT, Sapienza PJ, and Lee AL, Chemical shift imprint of intersubunit communication in a symmetric homodimer, Proc. Natl. Acad. Sci. U.S.A. (2016), 113, 9533-9538. (Commentary in same issue, pp. 9407-9409)
Sapienza PJ, Li L, Williams T, Lee AL, and Carter C, An ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures, ACS Chem Biol (2016), 11, 1661-1668.
Francis K, Sapienza PJ, Lee AL, and Kohen A, The effect of the protein mass modulation on human dihydrofolate reductase, Biochemistry (2016), 55, 1100-1106.
Sapienza PJ, Falk BT, and Lee AL, Bacterial thymidylate synthase binds two molecules of substrate and cofactor without cooperativity, JACS (2015), 137, 14260-14263.
Lee AL, Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95, Biophysical Reviews (2015), 7, 217-226.Sapienza PJ and Lee AL, Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analog, Biomolecular NMR Assignments (2014), 8, 195-199.
- UNC Eshelman School of Pharmacy
- UNC Department of Biochemistry and Biophysics
- UNC Biological & Biomedical Sciences Program (BBSP)
- UNC Biophysics Program
- Macromolecular Interactions Facility
- UNC Eshelman School of Pharmacy NMR Laboratory
- UNC Biomolecular NMR Laboratory
- NMR Information Server
- Biological Magnetic Resonance Data Bank (BMRB)
- Protein Data Bank PDB
Michael “Sparky” Clarkson (2005)
Josh Boyer (2009)
Randy Mauldin (2009)
Matthew Whitley (2010)
Mary Carroll/Harner (2011)
Jun Zhang (2011)
Tony Law (2011)
Leanna McDonald (2013)
Brad Falk (2017)
Ernesto Fuentes (2005)
Paul Sapienza (2011)
Chad Petit (2012)
Maria McGresham (2016)