Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

Ziad Ibrahim,Martine Moulin,Henry S Kim,Bruno Franzetti,Michael Härtlein,Anne Martel,Frank Gabel

doi:10.1038/srep40948

Abstract

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.

Highlights

AAA+(ATPases Associated with diverse cellular Activities) unfoldase complexes represent a large family of protein remodelling machines which play an essential role in the clearance of damaged or misfolded proteins as well as in specific regulatory tasks, including the degradation of cell-cycle components, transcription factors and metabolic enzymes[1,2,3]
The results show that ATPγS blocks GFP substrate (GFPssrA) unfolding by PAN since the Small-angle neutron scattering (SANS) profiles remained identical over 50 min at 55 °C (Supplementary Fig. S4)
Due to the high performance of the D22 diffractometer at the Institut Laue-Langevin neutron facility, combined with perdeuteration, it was possible to run the experiment under modest sample requirements (small volumes (~300 μl), low protein concentrations (~2 mg/ml) of relatively small molecules (GFPssrA, ~28 kDa)) and yet reach short exposure times (30 seconds) and obtain structural information during the active unfolding process

Summary

Introduction

AAA+(ATPases Associated with diverse cellular Activities) unfoldase complexes represent a large family of protein remodelling machines which play an essential role in the clearance of damaged or misfolded proteins as well as in specific regulatory tasks, including the degradation of cell-cycle components, transcription factors and metabolic enzymes[1,2,3]. Energy-dependent proteases are found across the three domains of life: bacteria contain multiple AAA+proteases (ClpXP, ClpAP, HslUV, FtsH and Mpa:20S), archaea have two known cytoplasmic AAA+proteases (PAN:20S and Cdc48:20S) and, in eukaryotes, a large part of cytosolic proteins are degraded by the 19S:20S system forming the 26S proteasome[2]. Other biophysical methods, including single-molecule optical trapping[13] or Förster resonance energy transfer (FRET)[14] have been applied to monitor unfolding by AAA+enzymes While these techniques provide accurate kinetic data and working models, direct structural information on AAA+. High (and low) resolution structural studies, carried out on different AAA+

Methods

Results

Conclusion