Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

Cell Rep. 2019 Jun 18;27(12):3433-3446.e4. doi: 10.1016/j.celrep.2019.05.075.

Abstract

AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.

Keywords: AAA+; Hsp100; chaperone; cryo-EM; protein disaggregation; protein unfolding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AAA Domain / genetics
  • ATPases Associated with Diverse Cellular Activities / chemistry
  • ATPases Associated with Diverse Cellular Activities / metabolism*
  • Adenosine Triphosphate / metabolism*
  • Cryoelectron Microscopy
  • Endopeptidase Clp / chemistry*
  • Endopeptidase Clp / genetics
  • Endopeptidase Clp / metabolism*
  • Endopeptidase Clp / ultrastructure
  • Escherichia coli / chemistry
  • Escherichia coli / enzymology
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Escherichia coli Proteins / ultrastructure
  • Heat-Shock Proteins / chemistry*
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism*
  • Heat-Shock Proteins / ultrastructure
  • Models, Molecular
  • Mutation
  • Protein Binding
  • Protein Domains / genetics

Substances

  • Escherichia coli Proteins
  • Heat-Shock Proteins
  • Adenosine Triphosphate
  • Endopeptidase Clp
  • ClpB protein, E coli
  • ATPases Associated with Diverse Cellular Activities