Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum

Mol Microbiol. 2021 Sep;116(3):943-956. doi: 10.1111/mmi.14781. Epub 2021 Jul 17.


Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

Keywords: archaea; cell membrane structures; circadian clocks; locomotion; phosphorylation; single-molecule imaging.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Circadian Clocks
  • Circadian Rhythm Signaling Peptides and Proteins / genetics*
  • Circadian Rhythm Signaling Peptides and Proteins / metabolism*
  • Movement*
  • Mutagenesis, Insertional / methods
  • Phosphorylation
  • Pyrococcus furiosus / physiology*
  • Sulfolobus acidocaldarius / physiology*


  • Archaeal Proteins
  • Circadian Rhythm Signaling Peptides and Proteins
  • Adenosine Triphosphatases