Hydrogen exchange solvent protection by an ATP analogue reveals conformational changes in ERK2 upon activation.
Structural and kinetic studies have provided extensive information about the molecular mechanisms of kinase activation by phosphorylation. However, it is still unclear how changes in protein dynamics and flexibility contribute to catalytic function. Mass spectrometry was used to probe changes in hydrogen/deuterium exchange in the MAP kinase, ERK2, in the presence and absence of the ATP analogue, AMP-PNP. In both active and inactive forms of ERK2, protection from hydrogen exchange by AMP-PNP binding was observed within conserved ATP binding motifs in the N-terminal lobe, which are known to directly interact with nucleotide in various protein kinases. In contrast, higher protection from exchange by AMP-PNP was observed in active ERK2 compared to inactive ERK2, in a region corresponding to the conserved DFG motif, which is located in the C-terminal lobe and coordinates Mg2+ at the catalytic site. Thus, AMP-PNP binding simultaneously protects residues within the N and C terminus in the active form of ERK2, but not the inactive form. This demonstrates that ERK2 binds nucleotide in two modes, in which active ERK2 adopts a closed conformation following nucleotide binding in solution, while inactive ERK2 adopts an open conformation. The finding provides novel evidence that phosphorylation of ERK2 facilitates interdomain closure, allowing proper orientation between ATP and substrate to facilitate phosphoryl transfer.
Lee
Thomas
T
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Hoofnagle
Andrew N
AN
Resing
Katheryn A
KA
Ahn
Natalie G
NG
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D006859:Hydrogen; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D012997:Solvents
D012997:Solvents; D006859:Hydrogen; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1