Solution NMR Spectroscopy for the Study of Enzyme Allostery

Lisi, George P.; Loria, J. Patrick

doi:10.1021/acs.chemrev.5b00541

Cited by 111 publications

(98 citation statements)

References 327 publications

(949 reference statements)

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“…In the MWC system, ligand binding causes a concerted shift in both subunits, where both lig 1 peaks would coincide with the lig 2 peak. Alternatively, one might expect to observe two possible linear triplet patterns, one where one of the lig 1 peaks coincides with either the lig 2 or lig 0 peaks and the other is partially shifted toward lig 2 , or one where both lig 1 peaks are partially shifted toward lig 2 (Fig. 6 C and D) (12).…”

Section: Discussionmentioning

confidence: 99%

“…Resolving the dUMP 1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP 1 peaks are found beyond the limits set by apo and dUMP 2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.…”

mentioning

confidence: 97%

“…Even though broadly recognized, longrange communication is not well understood mechanistically (1)(2)(3)(4). Although there have been numerous strategies to reveal the structural and dynamic underpinnings of allostery, oddly these strategies have largely focused on complex oligomeric or, alternatively, on small monomeric allosteric proteins.…”

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confidence: 99%

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Chemical shift imprint of intersubunit communication in a symmetric homodimer

Falk

Sapienza

Lee

2016

Proc. Natl. Acad. Sci. U.S.A.

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Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig 1 ") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig 1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig 1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP 1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP 1 peaks are found beyond the limits set by apo and dUMP 2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.A llosteric regulation in proteins is a ubiquitous mechanism for controlling cellular behavior and an attractive strategy for therapeutic development. Even though broadly recognized, longrange communication is not well understood mechanistically (1-4). Although there have been numerous strategies to reveal the structural and dynamic underpinnings of allostery, oddly these strategies have largely focused on complex oligomeric or, alternatively, on small monomeric allosteric proteins. A likely more straightforward approach is to study allosteric mechanisms using simple symmetric homodimeric proteins, which would allow for answering the basic and general question of how the occurrence of an event in one subunit is communicated to another subunit, as occurs in classical multisubunit allosteric proteins. Given the large number of homodimeric proteins involved in cellular regulation (such as growth factors, cytokines, kinases, G protein-coupled receptors, transcription factors, and metabolic proteins), insights into intersubunit communication should be widely beneficial (5).As a key step toward a broad understanding of allosteric mechanisms, it will be important to observe how binding a ligand in one subunit is communicated to a second subunit, even in the absence of conformational change. Although this communication is straightforwar...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 97%

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Chemical shift imprint of intersubunit communication in a symmetric homodimer

Falk

Sapienza

Lee

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Spin-relaxation rates, especially those of transverse ( R 2 ) magnetization, have been particularly useful to characterize the rate of conformational exchange between such states on the millisecond time-scale, further providing quantifiable information on the equilibrium site populations ( p A and p B ). In recent years, the relaxation-compensated Carr-Purcell-Meiboom-Gill (rcCPMG) and R 1ρ experiments have been extensively used to characterize functionally relevant conformational exchange at specific local and/or global atomic sites within enzymes, specifically because they sample the millisecond time-scale of catalysis ( k cat ) in most enzyme systems (Kleckner & Foster, 2011; Lisi & Loria, 2016; Palmer, 2015). The rcCPMG experiment has been the method of choice to probe a two-site chemical exchange experienced by 1 H- 15 N backbone amide vectors ( 15 N-CPMG), providing a precise atomic-scale measure of the millisecond dynamics experienced by nearly every residue on a given enzyme.…”

Section: Ascertaining Conformational Sub-states and Populations Frmentioning

confidence: 99%

Conformational Sub-states and Populations in Enzyme Catalysis

Agarwal

Doucet

Chennubhotla

et al. 2016

Methods in Enzymology

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Enzyme function involves substrate and cofactor binding, precise positioning of reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme-substrate complex allows reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surrounding environment. Areas of low energy along the landscape form conformational substates, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations is challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling and nuclear magnetic resonance (NMR) have been developed for quantitative characterization of these substates and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.

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“…allostery | NMR | community networks | millisecond motions T he underlying principles of allosteric regulation have been a focal point of enzymology and structural biology for decades as studies of allostery have evolved from two phenomenological models (1,2) to recognize structural and conformational ensembles that define a broad range of enzymatic states (3)(4)(5)(6). As a result, a great deal of emphasis has been placed on understanding dynamic contributions to allostery (7)(8)(9)(10)(11)(12) and factors that enable small fluctuations in local conformations of enzymes to propagate information over large distances.…”

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confidence: 99%

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

Lisi

East

Batista

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand, N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.allostery | NMR | community networks | millisecond motions T he underlying principles of allosteric regulation have been a focal point of enzymology and structural biology for decades as studies of allostery have evolved from two phenomenological models (1, 2) to recognize structural and conformational ensembles that define a broad range of enzymatic states (3-6). As a result, a great deal of emphasis has been placed on understanding dynamic contributions to allostery (7-12) and factors that enable small fluctuations in local conformations of enzymes to propagate information over large distances. A well-developed understanding of dynamic allostery opens up numerous avenues for insight into protein engineering (13,14), drug design (15), and mechanistic biochemistry (16)(17)(18)(19)(20)(21)(22). To establish universal principles for relevant enzymes, however, a link between dynamics and function must be made.The heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS) plays a crucial role in amino acid and purine biosynthesis in bacteria and other microorganisms by sequentially catalyzing the hydrolysis of glutamine (Gln) in its HisH subunit and the cyclization of the substrate and allosteric effector N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) in its HisF subunit (Scheme 1). IGPS is a V-type allosteric enzyme that is inactive unless PRFAR is bound, and communication between the active and effector sites is driven by the enhancement of conformational flexibility, namely, fluctuations taking place on the millisecond timescale (22-24). Based on NMR and computational evidence, these motions are believed to propagate from the PRFAR binding site to the glutaminase binding site, thereby disrupting a hydrogen bond between V51 and P10 of HisH. This H bond prevents formation of the oxyanion hole site necessary for glutamine hydrolysis, and its disruption allows IG...

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Solution NMR Spectroscopy for the Study of Enzyme Allostery

Cited by 111 publications

References 327 publications

Chemical shift imprint of intersubunit communication in a symmetric homodimer

Chemical shift imprint of intersubunit communication in a symmetric homodimer

Conformational Sub-states and Populations in Enzyme Catalysis

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

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