ATP hydrolysis inactivating Walker B mutation perturbs E. coli ClpA self-assembly energetics in the absence of nucleotide

Academic Article

Abstract

  • © 2018 E. coli ClpA is an AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins for the purposes of proper proteome maintenance. Biologically active ClpA hexamers contain two nucleotide binding domains (NBD) per protomer, D1 and D2. Despite extensive study, complete understanding of how the twelve NBDs within a ClpA hexamer coordinate ATP binding and hydrolysis to polypeptide translocation is currently lacking. To examine nucleotide binding and coordination at D1 and D2, ClpA Walker B variants deficient in ATP hydrolysis at one or both NBDs have been employed in various studies. In the presence of ATP, it is widely assumed that ClpA Walker B variants are entirely hexameric. However, a thermodynamically rigorous examination of the self-assembly mechanism has not been obtained. Differences in the assembly due to the mutation can be misattributed to the active NBD, leading to potential misinterpretations of kinetic studies. Here we use sedimentation velocity studies to quantitatively examine the self-assembly mechanism of ClpA Walker B variants deficient in ATP hydrolysis at D1, D2, and both NBDs. We found that the Walker B mutations had clear, if modest, effects on the assembly. Most notably, the Walker B mutation stabilizes the population of a larger oligomer in the absence of nucleotide, that is not present for analogous concentrations of wild type ClpA. Our results indicate that Walker B mutants, widely used in studies of AAA+ family proteins, require additional characterization as the mutation affects not only ATP hydrolysis, but also the ligand linked assembly of these complexes. This linkage must be considered in investigations of unfolding or other ATP dependent functions.
  • Published In

    Digital Object Identifier (doi)

    Pubmed Id

  • 2676189
  • Author List

  • Duran EC; Lucius AL
  • Start Page

  • 6
  • End Page

  • 14
  • Volume

  • 242