Structural heterogeneity of uncrosslinked FtsH-HflKC assemblies includes variable FtsH occupancy and a distinct wild-type FtsH architecture

The AAA+ metalloprotease FtsH collaborates with HflK and HflC to create the FtsH-HflKC complex (FHC), which works to maintain membrane protein quality in Escherichia coli. Recent structural investigations have shown that HflKC can have asymmetrical architectures and variable FtsH attachments, differing from the previously observed symmetric four-hexamer cage.

The team examined wild-type, uncrosslinked FHC co-produced from a single multicistronic plasmid and extracted in lauryl maltose neopentyl glycol (LMNG) by means of single-particle cryo-EM, SEC-SAXS, and biochemical activity tests. Two-dimensional analysis uncovered HflKC particles accompanied by one or two FtsH hexamers, as well as a unique collection of single FtsH particles.

Reconstruction of the FtsH cytosolic domain, focusing on the wild-type version, provided a structural perspective without incorporating any stabilizing modifications or a nucleotide analogue. The reconstruction showcased the recognition of the AAA+ and protease rings in a stacked configuration, albeit with a more outward-splayed AAA+ ring compared to the stabilized reference; the protease ring, however, remained largely consistent overall.

By using 3D variability analysis in C1, it was found that HflKC demonstrated significant conformational variability, ranging from open-like to more compact scaffold geometries. Solubilizing a selected LMNG fraction also yielded a compact, anisotropic envelope with an estimated Dmax of 110-115 angstroms; though, this entity could not be identified as an intact FHC with a specific stoichiometry.

Additionally, at the tested conditions, the FHC preparation exhibited lower apparent ATPase and proteolytic activities than the isolated FtsH. These discoveries emphasize the compositional and conformational variability of uncrosslinked FHC and expose a more outwardly splayed AAA+ arrangement in wild-type FtsH in comparison to the stabilized reference structure.