Insight into the role of dynamics in the conformational switch of the small GTP-binding protein Arf1. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Journal of Biological Chemistry Année : 2010

Insight into the role of dynamics in the conformational switch of the small GTP-binding protein Arf1.

Résumé

Activation of the small GTP-binding protein Arf1, a major regulator of cellular traffic, follows an ordered sequence of structural events, which have been pictured by crystallographic snapshots. Combined with biochemical analysis, these data lead to a model of Arf1 activation, in which opening of its N-terminal helix first translocates Arf1-GDP to membranes, where it is then secured by a register shift of the interswitch β-strands, before GDP is eventually exchanged for GTP. However, how Arf1 rearranges its central β-sheet, an event that involves the loss and re-formation of H-bonds deep within the protein core, is not explained by available structural data. Here, we used Δ17Arf1, in which the N-terminal helix has been deleted, to address this issue by NMR structural and dynamics analysis. We first completed the assignment of Δ17Arf1 bound to GDP, GTP, and GTPγS and established that NMR data are fully consistent with the crystal structures of Arf1-GDP and Δ17Arf1-GTP. Our assignments allowed us to analyze the kinetics of both protein conformational transitions and nucleotide exchange by real-time NMR. Analysis of the dynamics over a very large range of timescale by (15)N relaxation, CPMG relaxation dispersion and H/D exchange reveals that while Δ17Arf1-GTP and full-length Arf1-GDP dynamics is restricted to localized fast motions, Δ17Arf1-GDP features unique intermediate and slow motions in the interswitch region. Altogether, the NMR data bring insight into how that membrane-bound Arf1-GDP, which is mimicked by the truncation of the N-terminal helix, acquires internal motions that enable the toggle of the interswitch.

Domaines

Chimie organique

Dates et versions

hal-00551779 , version 1 (04-01-2011)

Identifiants

Citer

Vanessa Buosi, Jean-Pierre Placial, Jean-Louis Leroy, Jacqueline Cherfils, Éric Guittet, et al.. Insight into the role of dynamics in the conformational switch of the small GTP-binding protein Arf1.. Journal of Biological Chemistry, 2010, 285 (49), pp.37987-94. ⟨10.1074/jbc.M110.134445⟩. ⟨hal-00551779⟩
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