Conformational Dynamics Analysis Of Mek1 Using Hydrogen/Deuterium Exchange Mass Spectrometry

Background: Activation of mitogen-activated protein kinases (MAPKs) is regulated by a phosphorylation cascade comprising three kinases, MAPK kinase kinase (MAP3K), MAPK ki-nase (MAP2K), and MAPK. MAP2K1 and MAPK2K2, also known as MEK1 and MEK2, activate ERK1 and ERK2. The structure of the MAPK signaling cascade has been studied, but high -resolu-tion structural studies of MAP2Ks have often focused on kinase domains or docking sites, but not on full-length proteins. Objective: To understand the conformational dynamics of MEK1. Methods: Full-length MEK1 was purified from Escherichia coli (BL21), and its conformational dy-namics were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The ef-fects of ATP binding were examined by co-incubating MEK1 and adenylyl-imidodiphosphate (AM-P-PNP), a non-hydrolysable ATP analog. Results: MEK1 exhibited mixed EX1/EX2 HDX kinetics within the N-terminal tail through beta 1, alpha I, and the C-terminal helix. AMP-PNP binding was found to reduce conformational dynamics within the glycine-rich loop and regions near the DFG motif, along with the activation lip. Conclusion: We report for the first time that MEK1 has regions that slowly change its folded and unfolded states (mixed EX1/EX2 kinetics) and also report the conformational effects of ATP-bind -ing to MEK1.