Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2.

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Cooperative polarization of MCAM/CD146 and ERM family proteins in melanoma.

The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile ("3P"), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although copolarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.

Conformation Selection by ATP-competitive Inhibitors and Allosteric Communication in ERK2.

Activation of the extracellular signal regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named "L" and "R", where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Intermittent treatment of BRAFV600E melanoma cells delays resistance by adaptive resensitization to drug rechallenge.

Patients with melanoma receiving drugs targeting BRAFV600E and mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2) invariably develop resistance and face continued progression. Based on preclinical studies, intermittent treatment involving alternating periods of drug withdrawal and rechallenge has been proposed as a method to delay the onset of resistance. The beneficial effect of intermittent treatment has been attributed to drug addiction, where drug withdrawal reduces the viability of resistant cells due to MAP kinase pathway hyperactivation. However, the mechanistic basis of the intermittent effect is incompletely understood. We show that intermittent treatment with the BRAFV600E inhibitor, LGX818/encorafenib, suppresses growth compared with continuous treatment in human melanoma cells engineered to express BRAFV600E, p61-BRAFV600E, or MEK2C125 oncogenes. Analysis of the BRAFV600E-overexpressing cells shows that, while drug addiction clearly occurs, it fails to account for the advantageous effect of intermittent treatment. Instead, growth suppression is best explained by resensitization during periods of drug removal, followed by cell death after drug readdition. Continuous treatment leads to transcriptional responses prominently associated with chemoresistance in melanoma. By contrast, cells treated intermittently reveal a subset of transcripts that reverse expression between successive cycles of drug removal and rechallenge and include mediators of cell invasiveness and the epithelial-to-mesenchymal transition. These transcripts change during periods of drug removal by adaptive switching, rather than selection pressure. Resensitization occurs against a background of sustained expression of melanoma resistance genes, producing a transcriptome distinct from that of the initial drug-naive cell state. We conclude that phenotypic plasticity leading to drug resensitization can underlie the beneficial effect of intermittent treatment.

Dynamic equilibria in protein kinases.

Structural changes involved in protein kinase activation and ligand binding have been determined from a wealth of X-ray crystallographic evidence. Recent solution studies using NMR, EPR, HX-MS, and fluorescence techniques have deepened this understanding by highlighting the underlying energetics and dynamics of multistate conformational ensembles. This new research is showing how activation mechanisms and ligand binding alter the internal motions of kinases and enable allosteric coupling between distal regulatory regions and the active site.

Activation Loop Dynamics Are Coupled to Core Motions in Extracellular Signal-Regulated Kinase-2.

The activation loop segment in protein kinases is a common site for regulatory phosphorylation. In extracellular signal-regulated kinase 2 (ERK2), dual phosphorylation and conformational rearrangement of the activation loop accompany enzyme activation. X-ray structures show the active conformation to be stabilized by multiple ion pair interactions between phosphorylated threonine and tyrosine residues in the loop and six arginine residues in the kinase core. Despite the extensive salt bridge network, nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiments show that the phosphorylated activation loop is conformationally mobile on a microsecond to millisecond time scale. The dynamics of the loop match those of previously reported global exchange within the kinase core region and surrounding the catalytic site that have been found to facilitate productive nucleotide binding. Mutations in the core region that alter these global motions also alter the dynamics of the activation loop. Conversely, mutations in the activation loop perturb the global exchange within the kinase core. Together, these findings provide evidence for coupling between motions in the activation loop and those surrounding the catalytic site in the active state of the kinase. Thus, the activation loop segment in dual-phosphorylated ERK2 is not held statically in the active X-ray conformation but instead undergoes exchange between conformers separated by a small energetic barrier, serving as part of a dynamic allosteric network controlling nucleotide binding and catalytic function.

Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O-methyltransferase reaction.

Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor-acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954-7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15-20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

Kinase Activation by Small Conformational Changes.

Protein kinases (PKs) are allosteric enzymes that play an essential role in signal transduction by regulating a variety of key cellular processes. Most PKs suffer conformational rearrangements upon phosphorylation that strongly enhance the catalytic activity. Generally, it involves the movement of the phosphorylated loop toward the active site and the rotation of the whole C-terminal lobe. However, not all kinases undergo such a large configurational change: The MAPK extracellular signal-regulated protein kinases ERK1 and ERK2 achieve a 50 000 fold increase in kinase activity with only a small motion of the C-terminal region. In the present work, we used a combination of molecular simulation tools to characterize the conformational landscape of ERK2 in the active (phosphorylated) and inactive (unphosphorylated) states in solution in agreement with NMR experiments. We show that the chemical reaction barrier is strongly dependent on ATP conformation and that the "active" low-barrier configuration is subtly regulated by phosphorylation, which stabilizes a key salt bridge between the conserved Lys52 and Glu69 belonging to helix-C and promotes binding of a second Mg ion. Our study highlights that the on-off switch embedded in the kinase fold can be regulated by small, medium, and large conformational changes.

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2.

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Specificity of Phosphorylation Responses to Mitogen Activated Protein (MAP) Kinase Pathway Inhibitors in Melanoma Cells.

The BRAF-MKK1/2-ERK1/2 pathway is constitutively activated in response to oncogenic mutations of BRAF in many cancer types, including melanoma. Although small molecules that inhibit oncogenic BRAF and MAP kinase kinase (MKK)1/2 have been successful in clinical settings, resistance invariably develops. High affinity inhibitors of ERK1/2 have been shown in preclinical studies to bypass the resistance of melanoma and colon cancer cells to BRAF and MKK1/2 inhibitors, and are thus promising additions to current treatment protocols. But still unknown is how molecular responses to ERK1/2 inhibitors compare with inhibitors currently in clinical use. Here, we employ quantitative phosphoproteomics to evaluate changes in phosphorylation in response to the ERK inhibitors, SCH772984 and GDC0994, and compare these to the clinically used MKK1/2 inhibitor, trametinib. Combined with previous studies measuring phosphoproteomic responses to the MKK1/2 inhibitor, selumetinib, and the BRAF inhibitor, vemurafenib, the outcomes reveal key insights into pathway organization, phosphorylation specificity and off-target effects of these inhibitors. The results demonstrate linearity in signaling from BRAF to MKK1/2 and from MKK1/2 to ERK1/2. They identify likely targets of direct phosphorylation by ERK1/2, as well as inhibitor off-targets, including an off-target regulation of the p38α mitogen activated protein kinase (MAPK) pathway by the MKK1/2 inhibitor, trametinib, at concentrations used in the literature but higher than in vivo drug concentrations. In addition, several known phosphorylation targets of ERK1/2 are insensitive to MKK or ERK inhibitors, revealing variability in canonical pathway responses between different cell systems. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates.

Structure of histone-based chromatin in Archaea.

Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Rear-polarized Wnt5a-receptor-actin-myosin-polarity (WRAMP) structures promote the speed and persistence of directional cell migration.

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration.

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1.

Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication.

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4)2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones.

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication.

Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect.

p38ß Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261.

Many enzymes are self-regulated and can either inhibit or enhance their own catalytic activity. Enzymes that do both are extremely rare. Many protein kinases autoactivate by autophosphorylating specific sites at their activation loop and are inactivated by phosphatases. Although mitogen-activated protein kinases (MAPKs) are usually activated by dual phosphorylation catalyzed by MAPK kinases (MAPKKs), the MAPK p38ß is exceptional and is capable of self-activation by cis autophosphorylation of its activation loop residue T180. We discovered that p38ß also autophosphorylates in trans two previously unknown sites residing within a MAPK-specific structural element known as the MAPK insert: T241 and S261. Whereas phosphorylation of T180 evokes catalytic activity, phosphorylation of S261 reduces the activity of T180-phosphorylated p38ß, and phosphorylation of T241 reduces its autophosphorylation in trans Both phosphorylations do not affect the activity of dually phosphorylated p38ß. T241 of p38ß is found phosphorylated in vivo in bone and muscle tissues. In myogenic cell lines, phosphorylation of p38ß residue T241 is correlated with differentiation to myotubes. T241 and S261 are also autophosphorylated in intrinsically active variants of p38α, but in this protein, they probably play a different role. We conclude that p38ß is an unusual enzyme that automodulates its basal, MAPKK-independent activity by several autophosphorylation events, which enhance and suppress its catalytic activity.

Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation.

The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk's activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation.

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2.

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for (13)C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of (13)C(1)H3-labeled methyls by comparing distance information obtained from three-dimensional methyl-methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl-methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed "NOE-methyl cluster" onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV (13)C(1)H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.