Phosphorylation-dependent changes in structure and dynamics in ERK2 detected by SDSL and EPR.
Mitogen-activated protein kinases are regulated by occupancy at two phosphorylation sites near the active site cleft. Previous studies using hydrogen exchange to investigate the canonical mitogen-activated protein kinase, extracellular signal-regulated protein kinase-2, have shown that phosphorylation alters backbone conformational mobility >10 A distal to the site of phosphorylation, including decreased mobility within amino acids 102-105 and increased mobility within 108-109. To further describe changes after enzyme activation, site-directed spin labeling at amino acids 101, 105-109, 111, 112 and electron paramagnetic resonance spectroscopy were used to investigate this region. The anisotropic hyperfine splitting of the spin labels in glassy samples was unchanged by phosphorylation, consistent with previous crystallographic studies that indicate no structural change in this region. At positions 101, 111, and 112, the mobility of the spin label was unchanged by diphosphorylation, consistent with little or no conformational change. However, diphosphorylation caused small but significant changes in rotational diffusion rates at positions 105-108 and altered proportions of probe in a motionally constrained state at positions 105, 107, and 109. Thus, electron paramagnetic resonance indicates reproducible changes in nanosecond side-chain mobilities at specific residues within the interdomain region, far from the site of phosphorylation and conformational change.
Hoofnagle
Andrew N
AN
School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Stoner
James W
JW
Lee
Thomas
T
Eaton
Sandra S
SS
Ahn
Natalie G
NG
D019943:Amino Acid Substitution; D003545:Cysteine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004789:Enzyme Activation; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D013113:Spin Labels; D019950:Mitogen-Activated Protein Kinase 1; D003545:Cysteine