Fig. 7: Model for the preferred maturation pathway of SDHA.

Synthesis of the data suggests a preferred pathway for SDHA maturation in healthy cells. The compositions of SDHA-containing assembly intermediates that are formed during the CII biogenesis are shown as space-filling models in the center. Both the FAD and the dicarboxylate are shown superposed above each structure to help illustrate the maturation process, but these molecules are buried and shielded from solvent in the SDHA-AF2-AF4 and SDHA-AF4 structures. A ribbon diagram of SDHAF2 in each structure is shown at the bottom, and a diagram of SDHAF4 in each structure is shown at the top. In the first step, apo-SDHA binds FAD, dicarboxylate, and SDHAF2 to form the first metastable intermediate. In this process, the disordered termini of isolated SDHAF2 partially organize along the surface of SDHA but do not form secondary structure. This metastable SDHA-AF2 uses bound dicarboxylate as a cofactor to promote the covalent attachment of FAD, and form holo-SDHA-AF2. In the second step, SDHAF4 binds to form SDHA-AF2-AF4. Here, both termini of SDHAF2 further increase their organization. In addition, the C-terminal region of SDHAF4 becomes organized but has little secondary structure. Notably, the C-terminus of SDHAF4 occludes the dicarboxylate binding site, and dicarboxylate is released. In the third step, the additional organization of SDHAF4 displaces SDHAF2 and forms SDHA-AF4. The active site dicarboxylate-binding position remains occluded in this structure, although we note that the dicarboxylate malonate binds at a distal position in the structure. This distal site is of unknown relevance to the CII maturation process. Whether SHAF4 binds to another protein prior to or during its interaction with SDHA in cells remains unknown at this time. In addition, the handling steps of SDHA following this initial maturation process but prior to integration into functional CII (PDB ID 3SFD)⁷⁶ remains unknown at this time.