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

Abstract

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.

REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures

“…The deconvolved concentration profiles show a rise and fall of the dimer component, with a concomitant dip in the monomer, which resembles the profiles obtained by fitting scattering from crystal structure models in the original publication[39]. The P(r) functions are also physically reasonable, with single peaks that decay smoothly to zero as r approaches the maximum dimension.…”

“…First, we chose to analyze a stopped-flow mixing dataset from the soluble nucleotide binding domains (NBDs) of the membrane transport protein MsbA, which was recently published and deposited in a public database [39]. In the experiment, a solution with nucleotide-free NBD monomers was rapidly mixed with ATP, resulting in ligand binding and dimerization, followed by ATP hydrolysis and dissociation back to the monomeric state.…”

“…As a first example of time-resolved SAXS data, we chose a recently published stopped-flow mixing dataset [39]. In the experiment, a soluble nucleotide binding domain (NBD) construct (residues 330-581 of the ATP-binding cassette transporter MsbA) was mixed with Mg 2+ -ATP in a 1:1 (v/v) ratio (final concentrations 500 µM NBD and 450 µM ATP).…”

“…Model-free deconvolution of a time-resolved mixing dataset in REGALS. (a) Scattering profiles from a previously reported time-resolved mixing experiment with MsbA NBD and ATP[39] shown as Kratky plots (red to blue). The peak position shifts to q ∼ 0.07Å −1 before returning to q ∼ 0.08Å−1 (denoted by curved arrow), indicating a transient increase in size.…”

A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter

“…Here, we demonstrate that the rates of phosphate, ATP, and ADP release correlate with the OF-to-IF transition. In the future, single-molecule fluorescence resonance energy transfer (Catipovic et al, 2019), time-resolved small-angle X-ray scattering (Josts et al, 2019), and stopped-flow analyses (Robson et al, 2009) could provide further insights into turnover kinetics and rate-limiting steps.…”