Misfolding-Associated Exposure of Buried Residues in Mutant Sod1 Facilitates Binding to TRAF6

Abstract

Mutations of the protein Superoxide Dismutase 1 (SOD1) are implicated in a subset of Amyotrophic Lateral Sclerosis (ALS) cases. The C-terminal domain of TNF Receptor-Associated Factor 6 (TRAF6) is a newly discovered interactor of misfolded mutants of SOD1, but not wild-type SOD1. However no structural information is available yet to characterize how the proteins might directly interact. We sought to investigate possible mechanisms of this interaction through molecular dynamics (MD) and metadynamics simulations of a dimeric model system, coarse-grained using the AWSEM force field. We used direct MD simulations to predict possible binding poses by subjecting the trajectory to hierarchical clustering. Metadynamics simulations were used to deduce the preferred binding regions on the protein surfaces from the potential of the mean force in the spherical (θ, φ) orientation space around each protein. We found that the SOD1-interacting surface of TRAF6 is broadly distributed about the equatorial region of the beta barrel, while the TRAF6 interacting surface of SOD1 is relatively localized. When SOD1 is well-folded, the TRAF6 heterodimer interaction surface involves a co-option of the native SOD1 homodimer interface, while if SOD1 contains disordered loops IV and VII, as typically occurs in the absence of stabilizing Zn2+ ion binding, the disordered loops now partake in novel interactions with TRAF6. Using cDNA constructs, we expressed variants of TRAF6 with mutations in the predicted binding sites and found that TRAF6T475D no longer interacts with G93A and E100G mutants of SOD1 and has reduced interaction with SOD1A4V. These observations suggest a structural mechanism for the SOD1-TRAF6 interaction and may have implications for developing ALS therapeutics in the future.