Solution NMR Insights into Docking Interactions Involving Inactive ERK2
Open accessAbstract
The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the suboptimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and noncanonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the noncanonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.
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The quality of 2D [1H,15N] TROSY spectra of MAPKs is significantly different from one another, with often many fewer peaks detected than expected, despite their nearly identical 3D structures.
“Interestingly, the quality of the 2D [ 1 H, 15 N] TROSY spectra of MAPKs are significantly different from one another, with often many fewer peaks detected than expected, despite their nearly identical 3D structures.58,61,65”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [65]
Sequence-specific backbone assignment has been achieved for p38a (82%), activated pTpYp38a (56%), and ERK2 (65%), including key MAPK regulatory elements.
“The basis of any NMR spectroscopy analysis is the sequence-specific backbone assignment, which has been achieved for p38a 47,58,61 (82%), activated pTpYp38a 69 (56%) and ERK2 65 (65%) and include key MAPK regulatory elements, especially the KIM binding pockets (93%/95% for p38a/ERK2) the glycine-rich loops (100%/100% for p38a/ERK2) and the activation loops (83%/none for p38a/ERK2).”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [65]
Differential engagement of the U_A and U_B pockets in ERK2 by ERK-specific scaffolds (ETS, PEA-15) and substrates (Elk-1) has also been observed.
“Differential engagement of these pockets in ERK2 by ERK-specific scaffolds (ETS, PAE-15) and substrates (Elk-1) have also been observed.65”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [65]
The adjacent residue in ERK has also been shown to experience chemical shifts upon KIM peptide binding.
“The adjacent residue in ERK has also been shown to experience chemical shifts upon KIM peptide binding.65”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [65]
Differences in the NMR spectra of MAPKs (p38a > ERK2 > JNK1) provide the first indications that dynamics likely plays a key role in their biological function.
“The latter point seems to be especially critical for MAPKs and the differences in their NMR spectra (spectra quality of MAPKs tested by the authors is p38a>ERK2JNK1) 58,61,65 are providing the first indications that dynamics likely plays a key role in their biological function.”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [65]
PEA-15 KIM binding is mediated primarily by the hydrophobic binding pockets of the KIM binding groove with very little interaction at the electrostatic (CD) sites.
“Subsequently, NMR CSP studies of Piserchio et al., 65 which probed the interaction of PEA-15 on ERK2, revealed that PEA-15 KIM binding is mediated primarily by the hydrophobic binding pockets of the KIM binding groove with very little interaction at the electrostatic (CD) sites.”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [65]
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References 60 · Cited by 41
- Authors
- Piserchio, A., Warthaka, M., Devkota, A. K., Kaoud, T. S., Lee, S., Abramczyk, O., Ren, P., Dalby, K. N., & Ghose, R.
- Affiliations
- City College of New York (US)
- The Graduate Center, CUNY (US)
- Corresponding author
- Dalby, Kevin N.
- Ghose, Ranajeet
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2011-04-19
- Volume
- 50
- Issue
- 18
- Pages
- 3660-3672
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- PMC
- PMC3103835
- OpenAlex
- W2043920244
- Cited by
- 41
- References
- 60
- Citations by year
- 2025: 2 · 2023: 3 · 2022: 1 · 2021: 1 · 2020: 2 · 2019: 4 · 2018: 1 · 2017: 2 · 2016: 3 · 2015: 4 · 2014: 2 · 2013: 4 · 2012: 8
- FWCI
- 2.33
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (GM 059802, R01GM059802, R01GM084278, GM079686, GM084278, R01GM079686)
- National Center for Research Resources (5G12-RR03060, G12RR003060)
- National Institute on Minority Health and Health Disparities (G12MD007603)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Keywords
- ERK2, mitogen-activated protein kinase, chemical shift perturbation, allosteric regulation, gatekeeper residue, signal transduction, backbone assignment
- MeSH terms
- Allosteric Site, Animals, Binding Sites, Escherichia coli, Humans, Molecular Conformation, Magnetic Resonance Spectroscopy, Peptides, Protein Binding, Models, Statistical, Protein Structure, Tertiary, Crystallography, X-Ray, Mitogen-Activated Protein Kinase 1, Amino Acid Motifs, MAP Kinase Signaling System, Extracellular Signal-Regulated MAP Kinases, Rats
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