Probing Slow Dynamics in High Molecular Weight Proteins by Methyl-TROSY NMR Spectroscopy:  Application to a 723-Residue Enzyme

Korzhnev, Dmitry M.; Kloiber, Karin; Kanelis, Voula; Tugarinov, Vitali; Kay, Lewis E.

doi:10.1021/ja039587i

Cited by 225 publications

(307 citation statements)

References 35 publications

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“…No evidence for dynamics was indicated when R ex ≈ 0 s −1 . For individual ILV methyl groups, the dispersion curves at 600, 800, and 900 MHz were fit to a two-state exchange model (A ⇌ B), yielding exchange rate constants (k ex = k AB + k BA ) (17,18). Under optimal conditions, the populations (p A and p B ) and the 13 C chemical shift differences between the two states, jΔω 13 Cj (in Hz, or jΔδ CPMG 13 Cj in ppm), were obtained for individual methyl groups (17,18).…”

Section: Resultsmentioning

confidence: 99%

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Phosphorylation releases constraints to domain motion in ERK2

Xiao¹,

Lee

Latham³

et al. 2014

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

143

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Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR 13 C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with k ex ≈ 300 s −1 (k AB ≈ 240 s −1 /k BA ≈ 60 s −1 ) and p A /p B ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N-and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of k ex ≈ 500 s −1 (k AB ≈ 15 s −1 /k BA ≈ 485 s −1 ) and p A /p B ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.T he MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling and a model for protein kinase activation mechanisms (1). ERK2 can be activated by MAP kinase kinases 1 and 2 (MKK1 and 2) through dual phosphorylation of Thr and Tyr residues located at the activation loop (Thr183 and Tyr185, numbered in rat ERK2) (1, 2). Phosphorylation at both sites is required for kinase activation, resulting in increased phosphoryl transfer rate and enhanced affinity for ATP and substrate (3).Conformational changes accompanying the activation of ERK2 have been documented by X-ray structures of the inactive, unphosphorylated (0P-ERK2) and the active, dual-phosphorylated (2P-ERK2) forms (4, 5). Phosphorylation rearranges the activation loop, leading to new ion-pair interactions between phosphoThr and phospho-Tyr residues and basic residues in the N-and C-terminal domains of the kinase core structure. This leads to a repositioning of active site residues surrounding the catalytic base, enabling recognition of the Ser/Thr-Pro sequence motif at phosphorylation sites and exposing a recognition site for interactions with docking sequences in substrates and scaffolds (6).Less is known about how changes in internal motions contribute to kinase activation. Previous studies using hydrogenexchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7-9) led to a model where conformational mobility at the hinge linking the N-and C-terminal domains is increased by phosph...

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Section: Resultsmentioning

confidence: 99%

“…This is typically plotted as a relaxation dispersion curve, which can be fit to a two-state conformational exchange process (e.g., A ⇌ B interconversion). Fitting extracts the populations and the exchange rates between states, thus reflecting the thermodynamics and kinetics of the system (17,18).…”

mentioning

confidence: 99%

Phosphorylation releases constraints to domain motion in ERK2

Xiao¹,

Lee

Latham³

et al. 2014

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

143

View full text Add to dashboard Cite

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“…To better understand the slow motions (micro-to-millisecond timescale) on PmrA in the presence of DNA, we performed a series of NMR relaxation dispersion experiments, which are capable of probing invisible, low-populated conformations in protein folding, ligand binding and allosteric regulation [24][25][26] . Because of limited solubility and high molecular weight of the PmrA-DNA complex, we used methyl-TROSY relaxation dispersion experiments 27 to probe the slow dynamics in this complex. Methyl groups experiencing slow timescale motions show changes in effective relaxation rate, R 2,eff , measured as a function of the frequency of refocusing pulses n CPMG ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…11). Measurements at both 600 and 850 MHz were fitted individually by use of the software relax 28 to a two-site exchange process (A2B), yielding populations (p A and p B ), exchange rate constants (k ex ¼ k AB þ k BA ) and the 1 H and 13 C chemical shift differences between the two states 27 (Supplementary Table 4). In total, 12 PmrA methyl groups exhibit slow dynamics on binding to DNA.…”

Section: Resultsmentioning

confidence: 99%

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

Hsiao¹

2016

Acta Cryst Sect A

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“…We performed a series of relaxation dispersion experiments (Bouvignies et al 2010;Hansen et al 2008a, b;Korzhnev et al 2004;Loria et al 1999) suited for detecting conformational states that are poorly populated and thus only transiently visited. The results indicated that two variants-CAP-T127L/ S128I and CAP-S62F-cAMP 2 -underwent an exchange between conformational states on the microsecond-tomillisecond timescale Kalodimos 2009, 2012) (Fig.…”

Section: Allosteric Regulation By Slow Dynamicsmentioning

confidence: 99%

The role of slow and fast protein motions in allosteric interactions

Tzeng

Kalodimos

2015

Biophys Rev

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Allostery is fundamentally thermodynamic in nature. Long-range communication in proteins may be mediated not only by changes in the mean conformation with enthalpic contribution but also by changes in dynamic fluctuations with entropic contribution. The important role of protein motions in mediating allosteric interactions has been established by NMR spectroscopy. By using CAP as a model system, we have shown how changes in protein structure and internal dynamics can allosterically regulate protein function and activity. The results indicate that changes in conformational entropy can give rise to binding enhancement, binding inhibition, or have no effect in the expected affinity, depending on the magnitude and sign of enthalpy-entropy compensation. Moreover, allosteric interactions can be regulated by the modulation a lowpopulated conformation states that serve as on-pathway intermediates for ligand binding. Taken together, the interplay between fast internal motions, which are intimately related to conformational entropy, and slow internal motions, which are related to poorly populated conformational states, can regulate protein activity in a way that cannot be predicted on the basis of the protein's ground-state structure.

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Probing Slow Dynamics in High Molecular Weight Proteins by Methyl-TROSY NMR Spectroscopy: Application to a 723-Residue Enzyme

Cited by 225 publications

References 35 publications

Phosphorylation releases constraints to domain motion in ERK2

Phosphorylation releases constraints to domain motion in ERK2

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

The role of slow and fast protein motions in allosteric interactions

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