Ile, Leu, and Val Methyl Assignments of the 723-Residue Malate Synthase G Using a New Labeling Strategy and Novel NMR Methods

Tugarinov, Vitali; Kay, Lewis E.

doi:10.1021/ja030345s

Cited by 343 publications

(460 citation statements)

References 50 publications

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“…Of these, 70 methyl peaks were assigned to 60 residues by combining data from site-directed mutagenesis, through-space 1 H-1 H nuclear Overhauser effects (NOEs) between 1 H, 13 C-labeled methyls analyzed using the X-ray structure of 0P-ERK2, and throughbond intraresidue (HMCM[CG]CBCA) experiments (Fig. S1) (4,5,22). Most ILV residues in the hydrophobic core of the kinase were assigned, including those in the structurally conserved α-helices (αC-αH) and β-strands (β1-β5, β7, β8) as well as nonconserved helices in the MAP kinase insert (α1L14, α2L14) and C terminus (αL16).…”

Section: Resultsmentioning

confidence: 99%

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%

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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“…In addition, assignment of resonances in regions with significant spectral overlap can be addressed with specific amino acid labeling [15][16][17] as well as compensating for the loss of proton-proton distance restraints due to deuteration. 18,19 Other methods, such as residual dipolar couplings 20 and paramagnetic relaxation enhancement, 21 have provided additional structural restraints crucial for large a-helical proteins (e.g., a-helical membrane proteins in detergent micelles). With these advances, six solution NMR b-barrel membrane protein structures (varying in the number of b-strands (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and molecular weight (16-31 kDa) have been determined.…”

Section: Introductionmentioning

confidence: 99%

“…18,19 Other methods, such as residual dipolar couplings 20 and paramagnetic relaxation enhancement, 21 have provided additional structural restraints crucial for large a-helical proteins (e.g., a-helical membrane proteins in detergent micelles). With these advances, six solution NMR b-barrel membrane protein structures (varying in the number of b-strands (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and molecular weight (16-31 kDa) have been determined. [1][2][3][4][5][6] In this study, the difficulties of assigning Opa 60 , a b-barrel membrane protein from Neisseria gonorrhoeae, 22 are outlined and strategies for circumventing these challenges are demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Fox

Columbus

2013

Protein Science

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Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of b-barrel membrane proteins using Opa 60 from Neisseria gonorrhoeae as a model system. Opa 60 is an eight-stranded b-barrel with long extracellular loops (~63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa 60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the b-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized b-barrel. The removal of the loop resonances significantly improved the assignment of the Opa 60 b-barrel region (97% of the resonances corresponding to the b-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of b-barrel membrane proteins.

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“…Assignment of BeF 3 À -activated PmrA in complex with 25-bp DNA involved TROSY-HNCA and HNN-NOESY 22 . The 'out-and-back' 3D HMCM(CG)CBCA experiment 21 was acquired on a 0.4-mM methyl-protonated {Ile(d1 13 15 N] sample of PmrA for assigning methyl groups. Stereospecific assignment of methyl groups of Leu and Val residues was as described 41 .…”

Section: Methodsmentioning

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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Ile, Leu, and Val Methyl Assignments of the 723-Residue Malate Synthase G Using a New Labeling Strategy and Novel NMR Methods

Cited by 343 publications

References 50 publications

Phosphorylation releases constraints to domain motion in ERK2

Phosphorylation releases constraints to domain motion in ERK2

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

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

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