Abstract
SKD3, also known as human CLPB, belongs to the AAA+ family of ATPases associated with various activities. Mutations in the SKD3/CLPB gene cause 3-methylglutaconic aciduria type VII and congenital neutropenia. SKD3 is upregulated in acute myeloid leukemia, where it contributes to anti-cancer drug resistance. SKD3 resides in the mitochondrial intermembrane space, where it forms ATP-dependent high-molecular weight complexes, but its biological function and mechanistic links to the clinical phenotypes are currently unknown. Using sedimentation equilibrium and dynamic light scattering, we show that SKD3 is monomeric at low protein concentration in the absence of nucleotides, but it forms oligomers at higher protein concentration or in the presence of adenine nucleotides. The apparent molecular weight of the nucleotide-bound SKD3 is consistent with self-association of 12 monomers. Image-class analysis and averaging from negative-stain electron microscopy (EM) of SKD3 in the ATP-bound state visualized cylinder-shaped particles with an open central channel along the cylinder axis. The dimensions of the EM-visualized particle suggest that the SKD3 dodecamer is formed by association of two hexameric rings. While hexameric structure has been often observed among AAA+ ATPases, a double-hexamer sandwich found for SKD3 appears uncommon within this protein family. A functional significance of the non-canonical structure of SKD3 remains to be determined.