Structural insights into GTP-coupled conformational changes in Mfn1 revealed by time-resolved transition metal ion FRET.

Outer mitochondrial membrane fusion is mediated by the mitofusin paralogs Mfn1 and Mfn2. Nucleotide-driven self-assembly and conformational changes are required for regulated membrane fusion activity, but the allosteric mechanisms remain enigmatic due to incomplete structural information. In this study, we investigate the GTP-coupled conformational dynamics of Mfn1 using time-resolved transition metal ion fluorescence resonance energy transfer (tmFRET). Using the minimal Mfn1 construct with the GTPase domain and helical bundle 1 (HB1) connected by Hinge 2, we engineered FRET pairs by incorporating a fluorescent noncanonical amino acid donor and a metal ion acceptor. For each state of the catalytic cycle, we measured tmFRET with fluorescence lifetimes and determined distance distributions, which can capture complex structural heterogeneity. Our distance measurements for the GDP-bound state matched predictions from the atomic resolution structure, establishing that the same open state, with GTPase and HB2 domains far apart, exists in solution. Our findings reveal that the previously reported transition state is not a single closed state in solution. Rather, the distance distributions indicate that the presence of GDP+Pi results in an equilibrium between the open and closed state. For the first time, we capture the GTP-bound and nucleotide free states of Mfn1. GTP binding favors the open state, revealing an unrecognized conformational change in the fusion mechanism driven by GTP hydrolysis. Finally, the conformation of the apo state is distinct from each nucleotide bound state. Our data reveal fundamental insights into the structures and energetics of GTP-driven conformational changes of Mfn1.