Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease

Abstract

The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

“…3b). This is consistent with the translocation direction of other substrate-engaged cryo-EM structures of ATPases, exhibiting the same characteristic right-handed spiral staircase arrangement of pore loops 1, as observed in our structure [42][43][44][45]47,57 . The pore loops 1 of chain A (Pex1) and E (Pex1) occupy the highest and the lowest position of the spiral (Fig.…”

Processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex

“…S4C). This substrate binding mode has not been observed previously for bacterial Lon ( 19 , 20 ). In the recent structure of human Lonp1, the bound substrate is similarly gripped by pore-loop-1 from five protomers; however, the bound polypeptide is engaged only by pore-loop-2 from two protomers ( 21 ).…”

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

“…Prior structural studies of substrate-translocating AAA+ proteins and Lon proteases are consistent with this model of hand-over-hand substrate translocation 13,14,16–18 . In this model, pore loop residues of ATP-bound subunits form a spiral arrangement wherein the lower-most subunit’s pore loop residue typically exists in an intermediary state.…”

“…In this model, pore loop residues of ATP-bound subunits form a spiral arrangement wherein the lower-most subunit's pore loop residue typically exists in an intermediary state. In concordance with the AAA+ organization of bacterial Lon proteases 13,18,19 , our substrate-translocating PIM1 structure consists of four descending ATPase domains (labeled here as ATP1-3, ATP4) and two ascending 'seam' subunits (ADP1-2). The ADP-bound subunits are found between the lowest and highest subunits of the substrate-bound spiral arrangement and ADP-bound subunit pore loop residues are indeed disengaged from substrate (Figure 1B, ADP1/ADP2).…”

“…Remarkably, data collection and image analysis yielded the structure of PIM1 in a hexameric, closed, substrate-translocating state at a reported resolution of ~3.2 Å. Notably, and in stark contrast to all prior cryo-EM studies of Lon proteases in other species 13,14,16–18 , we did not observe the presence of a fully ADP-bound substrate-free oligomer, typically defined by the AAA+ domains adopting an open, left-handed spiral organization. We note a high degree of background in our micrographs, which we believe to be monomeric PIM1 derived from biochemical instability of the hexamer or particle disruption at the air-water interface ( Supplementary Figure 2A ).…”