TY - JOUR

T1 - An NMR Study of a 300-kDa AAA+ Unfoldase

AU - Krüger, Georg

AU - Kirkpatrick, John

AU - Mahieu, Emilie

AU - Franzetti, Bruno

AU - Gabel, Frank

AU - Carlomagno, Teresa

N1 - Funding Information: This work was funded by the Deutsche Forschungsgemeinschaft through grant CA294/13-1 to T.C and by the Leverhulme Trust through a Leverhulme International Professorship to T.C. We thank Susanne zur Lage (HZI Braunschweig) for help with sample preparation.

PY - 2023/6/1

Y1 - 2023/6/1

N2 - AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

AB - AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

KW - AAA+ ATPase

KW - methyl NMR

KW - PAN

KW - unfoldase

UR - http://www.scopus.com/inward/record.url?scp=85165710380&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2023.167997

DO - 10.1016/j.jmb.2023.167997

M3 - Article

C2 - 37330287

AN - SCOPUS:85165710380

VL - 435

JO - Journal of molecular biology

JF - Journal of molecular biology

SN - 0022-2836

IS - 11

M1 - 167997

ER -