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Andrew Lee Lab

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lee_lab_image1We 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.

CheY_allosteric_network-300x254

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).

CM_ribbonsChorismate 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.

TS-cartoon-and-spectrum-245x300Most recently, we have been studying the 62 kD dimeric enzyme thymidylate synthase, or TS. This enzyme is metabolically critical, performing conversion of uridine monophosphate to thymidine monophosphate. TS is more complex than many of the enzymes studied to date by NMR as it has a multistate 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 nonreactivity to the other subunit. This is essentially a form of high negative cooperativity. Our NMR studies of TS will also focus on this intersubunit negative cooperativity and the structural and dynamic features associated with it. In summary, TS is a fascinating, large enzyme that it interesting from both the perspective of catalysis and allostery.

drug_binding_dynamicsMany proteins serve as drug receptors. In such cases the dynamics may contribute to drug-binding affinity or residence times, both of which are important metrics for efficacious drugs. From our NMR studies on dihydrofolate reductase (DHFR), we have observed that conformational dynamics can correlate with inhibitor dissociation rates, and we have even observed “dynamic ligands” that rapidly switch their conformations while bound to the protein. We remain interested in this general area.

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.

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.

Andrew L Lee

(919) 966-7821

drewlee@unc.edu

ACCEPTING DOCTORAL STUDENTS

Andrew Lee studies the role of conformational dynamics in protein function, conformational changes, enzyme catalysis, drug binding, and allostery. His laboratory uses a variety of biophysical and biochemical tools, especially NMR spectroscopy. NMR spectroscopy is a powerful approach that yields atomic-resolution molecular information and is uniquely sensitive to molecular fluctuations over a broad range of timescales.

Paul Sapienza

919-843-5442

sapienza@email.unc.edu

Paul Sapienza is a research assistant professor in the Division of Chemical Biology and Medicinal Chemistry at the UNC Eshelman School of Pharmacy. His research aims to further understanding of the role of dynamics in biomolecular recognition, enzymatic catalysis, and allostery. He uses nuclear magnetic resonance spectroscopy to study protein dynamics on multiple timescales, while other tools such as calorimetry, crystallography, and kinetics serve to link dynamics with function. He is focusing on thymidylate synthase as it is an enzyme with a multistep catalytic cycle, is a cancer drug target, and exhibits negative cooperativity (allostery).

Lab Alumni

PhD

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)

Postdoctoral

Ernesto Fuentes (2005)

Paul Sapienza (2011)

Chad Petit (2012)

Maria McGresham (2016)

The Lee lab is anticipating an opening for a postdoctoral position for NMR and other biophysical studies of the dynamics, structure, and solution behavior of a classically allosteric enzyme. Two projects in the lab focus on uncovering mechanisms of allosteric communication in enzyme homodimers in the 60-70 kDa range. Both project areas will involve advanced NMR spectroscopy for large proteins, including spin relaxation, as well as other biophysical methods (e.g. calorimetry), enzyme assays, protein conjugation using click chemistry, and recently developed “protein network” analysis techniques. Candidates are sought with expertise in any one or more of the areas including:  protein structural biology and biophysics, protein conjugation chemistry, enzymology and NMR spectroscopy. Interested candidates should send their CV to Dr. Lee (drewlee@unc.edu).