The goal of this proposal is to investigate the motional changes that occur in response^? protein kinase activation, and explore their relevance to enzyme function. Analysis of the MAP^kinase^ ERK2, by hydrogen exchange mass spectrometry (HX-MS) and other measurements reveal three situations in which enzyme perturbations involving (i) enzyme activation, (ii) allosteric communication between binding sites, and (iii) mutagenesis lead to localized changes in protein conformational mobility over long distances. The available evidence suggests a novel model for signal transduction control, in which the regulation of protein dynamics controls catalytic and allosteric functions in ERK2.
Specific aims i n this proposal will test this hypothesis, and examine behavior of related MAP kinases. Sp.
Aim 1 will analyze protein dynamics in ERK2 by HX-MS, NMR and FRET in order to determine the functional consequence of flexibility changes at the hinge that are induced by kinase phosphorylation and activation. Sp.
Aim 2 will test the hypothesis that MAP kinase docking motifs interact allosterically between their respective binding pockets, and explore the functional consequences of allosteric regulation, using isothermal calorimetry, HX-MS, and NMR. Sp.
Aim 3 will investigate the mechanism by which mutations in the N-terminal domain of ERK2 regulate flexibility at the activation lip, by site-directed mutagenesis, HX-MS and NMR. Sp.
Aim 4 will extend our HX-MS analyses to document activation-dependent conformational mobility in other protein kinases. Completion of these aims will provide a unique window for understanding how protein kinases have evolved to optimize function by controlling conformational mobility. Innovative technologies will be applied to this problem, including high field solution NMR and hydrogen exchange mass spectrometry. The experiments will: (i) document the regulation of internal motions in a large enzyme, (ii) demonstrate the role of protein motions in controlling kinase enzymatic function, (iii) improve our understanding of how protein motions are controlled over long distances, and (iv) document new mechanisms for regulation of signaling molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074134-04
Application #
7746483
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Gerratana, Barbara
Project Start
2007-01-01
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2011-12-31
Support Year
4
Fiscal Year
2010
Total Cost
$245,998
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Xiao, Yao; Warner, Lisa R; Latham, Michael P et al. (2015) 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. Biochemistry 54:4307-19
Xiao, Yao; Liddle, Jennifer C; Pardi, Arthur et al. (2015) Dynamics of protein kinases: insights from nuclear magnetic resonance. Acc Chem Res 48:1106-14
Sours, Kevin M; Xiao, Yao; Ahn, Natalie G (2014) Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility. J Mol Biol 426:1925-35
Xiao, Yao; Lee, Thomas; Latham, Michael Parker et al. (2014) Phosphorylation releases constraints to domain motion in ERK2. Proc Natl Acad Sci U S A 111:2506-11
Ring, Adam Y; Sours, Kevin M; Lee, Thomas et al. (2011) Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2. Int J Mass Spectrom 302:101-109
Oyeyemi, Olayinka A; Sours, Kevin M; Lee, Thomas et al. (2011) Comparative hydrogen-deuterium exchange for a mesophilic vs thermophilic dihydrofolate reductase at 25 °C: identification of a single active site region with enhanced flexibility in the mesophilic protein. Biochemistry 50:8251-60
Sours, Kevin M; Ahn, Natalie G (2010) Analysis of MAP kinases by hydrogen exchange mass spectrometry. Methods Mol Biol 661:239-55
Oyeyemi, Olayinka A; Sours, Kevin M; Lee, Thomas et al. (2010) Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency. Proc Natl Acad Sci U S A 107:10074-9
Sours, Kevin M; Kwok, Stan C; Rachidi, Thami et al. (2008) Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38alpha MAP kinase. J Mol Biol 379:1075-93