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Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering

2019; Elsevier BV; Volume: 28; Issue: 3 Linguagem: Inglês

10.1016/j.str.2019.12.001

1878-4186

Inokentijs Josts, Yunyun Gao, Diana C. F. Monteiro, Stephan Niebling, Julius Nitsche, Katharina Veith, Tobias Gräwert, Clément E. Blanchet, Martin A. Schroer, Nils Huse, Arwen R. Pearson, Dmitri I. Svergun, Henning Tidow,

Protein Structure and Dynamics

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.