University of Colorado
Conformational Dynamics in the Regulation of MAP Kinase, ERK2.
Degree: PhD, Chemistry & Biochemistry, 2015, University of Colorado
The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling. Aberrant up-regulation of ERK2 has been correlated with various diseases. ERK2 can be activated by MAP kinase kinases through dual phosphorylation at the activation loop. It remains a challenging question how changes in conformational dynamics contribute to kinase activation. NMR relaxation dispersion experiments were used to monitor changes in Ile, Leu, and Val (ILV) methyl motions in microsecond-millisecond timescale upon activation of ERK2. A structure-based procedure was developed to assign 13C1H3-labeled methyls, by comparing NMR distance constraints with the X-ray structure. This procedure yielded 60% of the methyl assignments in inactive and active forms of ILV 13C1H3-methyl labeled ERK2. In inactive ERK2, localized conformational dynamics was observed among methyls. Upon activation, the dynamics of assigned methyls in ERK2 were altered throughout the kinase core, including many residues in the catalytic pocket. The majority of methyls in active ERK2 fit to a single conformational exchange process, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance flexibility at the hinge region linking the N- and C-terminal domains, induced two-state conformational exchange throughout the kinase core. A mono-phospho-mimetic form of this mutant showed 25% of the dual-phosphorylated ERK2 activity. Thus, activation of ERK2 leads to a dramatic shift in conformational exchange, from a “tense” (T) state to a “relaxed” (R) state, likely through release of constraints at the hinge. To understand the effects on the conformational dynamics of ERK2 during catalysis and upon inhibitor binding, complexes of ERK2 with various ligands were formed. The binding of nucleotides and/or peptide substrates showed no significant perturbation to the T/R conformational equilibrium, with small enhancement of the T state population in active ERK2. In addition, differential conformational stabilization effects, which were not previously reported for ERK2, were observed upon the binding of different tight-binding inhibitors of ERK2. This thesis reports that ERK2 activation enhances microsecond-millisecond interconversion between conformers underlying different enzyme intermediates, thus linking protein dynamics to the catalytic cycle. The perturbations of conformational equilibrium by inhibitors reflect a novel allosteric mechanism in ERK2.
Advisors/Committee Members: Natalie G. Ahn, Arthur Pardi, Marcelo Sousa, Johannes Rudolph, Loren Hough.
Subjects/Keywords: ERK2; MAP kinase; aberrant up-regulation; interconversion; conformation exchange; Biochemistry; Cell Biology
to Zotero / EndNote / Reference
APA (6th Edition):
Xiao, Y. (2015). Conformational Dynamics in the Regulation of MAP Kinase, ERK2. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/168
Chicago Manual of Style (16th Edition):
Xiao, Yao. “Conformational Dynamics in the Regulation of MAP Kinase, ERK2.” 2015. Doctoral Dissertation, University of Colorado. Accessed April 13, 2021.
MLA Handbook (7th Edition):
Xiao, Yao. “Conformational Dynamics in the Regulation of MAP Kinase, ERK2.” 2015. Web. 13 Apr 2021.
Xiao Y. Conformational Dynamics in the Regulation of MAP Kinase, ERK2. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2021 Apr 13].
Available from: https://scholar.colorado.edu/chem_gradetds/168.
Council of Science Editors:
Xiao Y. Conformational Dynamics in the Regulation of MAP Kinase, ERK2. [Doctoral Dissertation]. University of Colorado; 2015. Available from: https://scholar.colorado.edu/chem_gradetds/168