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

Abstract

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.

Towards Elucidating the Rotary Mechanism of the Archaellum Machinery

“…The ~36° steps, on the other hand, were hypothesised to be either associated with the hypothetical presence of 9–10 monomers of the modulator ArlH in the motor complex (as observed by Chaudhury et al, 2016 ), or motor slippage. Recent evidence suggests that ArlH forms a hexamer in the motor ( Sousa Machado et al, 2021 ), indicating the 9–10 stoichiometry of ArlH observed in previous single particle cryoEM data ( Chaudhury et al, 2016 ) may have been an artefact of sample preparation. A more recent calculation indicated that the torque output of H. salinarum archaella is 160 pN.nm regardless of its load ( Iwata et al, 2019 ).…”

“…ArlI is the hexameric ATPase, which localises to the cytosolic side of the plasma membrane, probably owing to its interaction with the platform protein ArlJ, whose structure has not yet been determined ( Reindl et al, 2013 ). ArlH is an auto-kinase that interacts with ArlI, modulating its activity and perhaps controlling when the ATPase assembles or rotates the filament ( Chaudhury et al, 2016 ; Sousa Machado et al, 2021 ). The filament structure has been obtained from P. furiosus (5O4U; Daum et al, 2017 ), and the remaining structures from Sulfolobus acidocaldarius proteins 5TUG ( Tsai et al, 2020 ); 4IHQ ( Reindl et al, 2013 ); and 4YDS ( Chaudhury et al, 2016 ).…”

“…ArlH itself does not hydrolyse ATP ( Chaudhury et al, 2016 , 2018 ; Meshcheryakov and Wolf, 2016 ). Instead, ArlH exhibits auto-phosphorylation activity ( Sousa Machado et al, 2021 ). ArlH has been shown to interact with other archaellum motor components, including ArlI ( Chaudhury et al, 2016 , 2018 ; Sousa Machado et al, 2021 ), ArlX ( Banerjee et al, 2013 ; Chaudhury et al, 2016 ), and ArlCDE ( Li et al, 2020 ).…”

“…ArlH has been shown to interact with other archaellum motor components, including ArlI ( Chaudhury et al, 2016 , 2018 ; Sousa Machado et al, 2021 ), ArlX ( Banerjee et al, 2013 ; Chaudhury et al, 2016 ), and ArlCDE ( Li et al, 2020 ). Phosphorylation of ArlH seems to influence both its oligomerisation and how it interacts with ArlI: the interaction between ArlI and ArlH is strongest when ArlH is not phosphorylated, and under these circumstances, ArlH adopts a hexameric form ( Sousa Machado et al, 2021 ). When ArlH autophosphorylates, the hexameric oligomer disassembles from the ArlI/ArlH complex ( Sousa Machado et al, 2021 ).…”

“…Phosphorylation of ArlH seems to influence both its oligomerisation and how it interacts with ArlI: the interaction between ArlI and ArlH is strongest when ArlH is not phosphorylated, and under these circumstances, ArlH adopts a hexameric form ( Sousa Machado et al, 2021 ). When ArlH autophosphorylates, the hexameric oligomer disassembles from the ArlI/ArlH complex ( Sousa Machado et al, 2021 ). It has been hypothesised that this is the signal that switches the archaellum machinery from filament assembly to filament rotation.…”