Hydrogen Exchange Solvent Protection by an ATP Analogue Reveals Conformational Changes in ERK2 upon Activation

Lee, Thomas; Hoofnagle, Andrew N.; Resing, Katheryn A.; Ahn, Natalie G.

doi:10.1016/j.jmb.2005.08.029

Cited by 45 publications

(73 citation statements)

References 36 publications

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“…phorylation of the MAP kinase activation loop leads to substantial changes of the main chain flexibility all around the ATP binding site (12). Similar results were obtained for PKA: phosphorylation of the activation loop caused global stabilization of the active site (13).…”

supporting

confidence: 74%

“…This leads to destabilizing of the magnesium-binding loop, movement of the ␣C-helix out of the active site, destruction of the hydrophobic spine, and loosening of the N-lobe. This model provides an explanation of the protein kinase stabilization induced by phosphorylation (12)(13)(14). In addition, the involvement of the lysine-glutamate bond into the stabilization phenomenon can explain the results observed in the K 72 H mutant of PKA (13).…”

Section: Discussionmentioning

confidence: 72%

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Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

Kornev

Haste²,

Taylor³

et al. 2006

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

668

746

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The surface comparison of different serine-threonine and tyrosine kinases reveals a set of 30 residues whose spatial positions are highly conserved. The comparison between active and inactive conformations identified the residues whose positions are the most sensitive to activation. Based on these results, we propose a model of protein kinase activation. This model explains how the presence of a phosphate group in the activation loop determines the position of the catalytically important aspartate in the AspPhe-Gly motif. According to the model, the most important feature of the activation is a ''spine'' formation that is dynamically assembled in all active kinases. The spine is comprised of four hydrophobic residues that we detected in a set of 23 eukaryotic and prokaryotic kinases. It spans the molecule and plays a coordinating role in activated kinases. The spine is disordered in the inactive kinases and can explain how stabilization of the whole molecule is achieved upon phosphorylation.protein surface ͉ graph theory

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supporting

confidence: 74%

Section: Discussionmentioning

confidence: 72%

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

Kornev

Haste²,

Taylor³

et al. 2006

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

668

746

View full text Add to dashboard Cite

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“…Previous studies using hydrogenexchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7)(8)(9) led to a model where conformational mobility at the hinge linking the N-and C-terminal domains is increased by phosphorylation, therefore releasing constraints needed for activation. Such a model differs from other types of autoinhibitory mechanisms in protein kinases, which involve interactions with domains outside the kinase core (10,11).…”

mentioning

confidence: 99%

Phosphorylation releases constraints to domain motion in ERK2

Xiao¹,

Lee

Latham³

et al. 2014

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

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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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“…Many enzymes can be crystallized in more than one structural conformation, raising the possibility that the structures with lowered energy barrier may be directly associated with the conversion of substrate to product. Diverse solution methods (5)(6)(7)(8) have also shown that specific regions within the enzyme are in dynamic flux and that the binding of ligands can significantly modify the dynamics in unique ways (9)(10)(11). Although these phenomena are likely to be critical for catalysis, a significant dilemma facing structural biology is to correlate experimentally obtained ''frozen'' structures with a quantitative understanding of the functional mechanism.…”

mentioning

confidence: 99%

“…Despite the simplicity of the reaction catalyzed by these enzymes, the protein kinases are regulated in complex ways that may involve phosphorylation by other kinases (14), membrane and organelle localization through scaffolding proteins (15), and protein-protein and domain-domain interactions through regulator modules (16,17). In the last decade, a full appreciation for the conformational complexity of these enzymes has been developed (11,18). The essential kinase core has been crystallized in many different conformations (19)(20)(21) that are distinguished by rotations between the ATP and substrate-binding lobes and by movements in various secondary structural elements.…”

mentioning

confidence: 99%