title: Phosphorylation releases constraints to domain motion in ERK2.
abstract: 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 kex ≈ 300 s(-1) (kAB ≈ 240 s(-1)/kBA ≈ 60 s(-1)) and pA/pB ≈ 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 kex ≈ 500 s(-1) (kAB ≈ 15 s(-1)/kBA ≈ 485 s(-1)) and pA/pB ≈ 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.
authors.affiliation: Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, and BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309.
authors.forename: Yao
authors.initials: Y
authors.lastname: Xiao
authors.forename: Thomas
authors.initials: T
authors.lastname: Lee
authors.forename: Michael Parker
authors.initials: MP
authors.lastname: Latham
authors.forename: Lisa Rose
authors.initials: LR
authors.lastname: Warner
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanimoto
authors.forename: Arthur
authors.initials: A
authors.lastname: Pardi
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Ahn
mesh_terms: D000818:Animals; D004789:Enzyme Activation / Q000502:physiology*; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1