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

The AAA+ metalloprotease FtsH associates with HflK and HflC to form the megadalton FtsH-HflKC complex (FHC), which contributes to membrane-protein quality control in Escherichia coli . Recent structural studies have revealed asymmetric HflKC architectures and variable FtsH occupancy, contrasting with earlier crosslinked structures of a symmetric four-hexamer cage. Here, we analyzed wild-type, uncrosslinked FHC co-expressed from a single multicistronic plasmid and solubilized in lauryl maltose neopentyl glycol (LMNG) using single-particle cryo-EM, SEC-SAXS and biochemical activity assays. 2D classification identified HflKC-containing particles associated with one or two FtsH hexamers, together with a distinct population of isolated FtsH particles. Focused reconstruction of the FtsH cytosolic domain reached 5.32 Å resolution and provided a structural view of wild-type, uncrosslinked FtsH without stabilizing substitutions or an added nucleotide analogue. The reconstruction retained the characteristic stacked arrangement of the AAA+ and protease rings but showed a more laterally splayed AAA+ ring compared with the stabilized reference, while the protease ring remained broadly similar in overall envelope. A 9.63 Å HflKC consensus reconstruction and 3D variability analysis (3DVA) in C1, further revealed substantial conformational variability spanning open-like and more constricted scaffold geometries. SEC-SAXS of a selected later-eluting LMNG-solubilized fraction yielded a compact anisotropic envelope with an estimated D _max of ∼110-115 Å, although this species could not be assigned to a defined intact FHC stoichiometry. Under the conditions tested, the FHC preparation also showed lower apparent ATPase and proteolytic activities than isolated FtsH. Together, these findings highlight the compositional and conformational heterogeneity of uncrosslinked FHC and reveal a more laterally splayed AAA+ arrangement in wild-type FtsH compared with the stabilized reference structure.