Gαs slow conformational transition upon GTP binding and a novel Gαs regulator.

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the ß2-adrenergic receptor (ß2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gαs from ß2AR and Gßγ, as opposed to a slow closing of the Gαs α-helical domain (AHD). Yeast-two-hybrid screening using Gαs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gαs AHD.

Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade.

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein-coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange-mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

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 kinase (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-resolution 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 dynamics were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The effects of ATP binding were examined by co-incubating MEK1 and adenylyl-imidodiphosphate (AMP- PNP), a non-hydrolysable ATP analog. RESULTS: MEK1 exhibited mixed EX1/EX2 HDX kinetics within the N-terminal tail through ß1, αI, 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-binding to MEK1.