AIFM1 Protein Acts as Metabolic Hub by Binding Energy Enzyme AK2A in Mitochondria

Mitochondrial energy production depends on precisely coordinated protein interactions in the intermembrane space (IMS), yet many of these relationships remain poorly understood. AIFM1, a FAD-dependent oxidoreductase already known for roles in cell death and respiratory chain biogenesis, has long been suspected to influence energy metabolism beyond its established interaction with MIA40. This study set out to define AIFM1's full interaction network and characterize its functional significance.

The researchers built a high-confidence AIFM1 interactome using three complementary approaches: native immunoprecipitation with label-free proteomics, SILAC-based quantitative IP, and unbiased in vitro protein microchip profiling with purified recombinant proteins. Across all three methods, they consistently identified adenylate kinase 2 (AK2) and MICOS complex components (MIC27, MIC19, MIC60) alongside the known partner MIA40. Gel filtration confirmed that endogenous AK2 co-migrates with AIFM1 in a stable, long-lived complex that persists even after cycloheximide treatment.

A critical discovery was isoform specificity: only AK2A, not AK2B, interacts with AIFM1. The two isoforms differ only in their C-terminal seven amino acids, and isothermal titration calorimetry confirmed direct binding of AK2A to AIFM1 with a Kd of 437 nM and a 1:2 (AK2A:AIFM1) stoichiometry—comparable to the MIA40-AIFM1 interaction. The conserved residues K233, D234, V236, and F238 in AK2A's unique C-terminal region mediate binding.

High-resolution cryo-EM structures of the AIFM1 dimer alone, AIFM1-AK2A, and AIFM1-MIA40 complexes (resolved at 2.8, 2.6, and 2.4 Å respectively) revealed that both AK2A and MIA40 bind the same β-sheet in AIFM1's C-terminal domain via parallel β-strand complementation. Both interactions lock AIFM1 in its active dimeric conformation and enhance NADH oxidoreductase activity at physiological NADH concentrations. MIA40 additionally affects the cofactor-binding site, suggesting nuanced regulatory differences between the two interactors. Shared hydrophobic contacts—particularly involving AIFM1 residues V505, V507, Y347, F508, and Y560—anchor both binding partners.

Functionally, the AIFM1-AK2A interaction appears especially important during the metabolic transition from fermentative to oxidative respiration. By recruiting AK2A near ADP/ATP carriers in the IMS, AIFM1 may create localized hotspots of adenine nucleotide-metabolizing enzymes, optimizing the adenylate pool available for mitochondrial energy transduction. These findings position AIFM1 as a bioenergetic scaffold, not merely a redox enzyme, and help explain why AIFM1 loss so broadly disrupts mitochondrial function in tissues with high energy demand.