Abstract
Mitochondria serve as a central hub for cellular energy production, making protein homeostasis and maintenance within the organelle essential to prevent disease1. CLPB is a AAA+ protein whose function is to disaggregate and refold toxic protein aggregates within the mitochondrial intermembrane space (IMS) using energy gained from ATP hydrolysis2,3,5. Upon binding ATP, human CLPB self assembles into a hexameric ring-like structure via its nucleotide binding AAA+ domain and can further oligomerize into a dodecamer via self-interacting ankyrin repeat domains3,4. HAX1, an anti-apoptotic signaling protein located in the IMS, is a strong interactor of CLPB, and mutations in both proteins lead to severe congenital neutropenia6,7,8. Deletion of CLPB in cells leads to increased HAX1 insolubility9. HAX1 residues 125-130 are essential for CLPB binding and are predicted to bind the CLPB ankyrin domain, in a region near where CLPB dodecamerization is mediated8. Given this evidence, we hypothesized that HAX1 may modulate CLPB activity. We tested how HAX1 binding altered CLPB disaggregase and ATPase activities, and how addition of HAX1 influenced CLPB oligomeric state. We discovered that HAX1 is a positive modulator, stimulating CLPB rates of ATP hydrolysis and disaggregation. Further, HAX1 alters CLPB oligomeric state, suggesting a model by which HAX1 binding regulates CLPB activity through modulation of CLPB oligomers.