Time-resolved tmFRET reveals GTP-coupled conformational changes in Mfn1.

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 HB1 domains far apart, exists in solution. Our data reveal that the transition state is not a single closed state with HB1 stably contacting the GTPase domain. Rather, the distance distributions indicate that the presence of GDP + Pi results in an equilibrium between the open and closed states. We also captured the GTP-bound and nucleotide-free states of Mfn1. GTP binding favors the open state, and the conformation of the apo state is distinct from any nucleotide-bound state. Together, these findings redefine our understanding of GTP-driven conformational dynamics in Mfn1, demonstrating an unexpected conformational reversal in a single catalytic cycle and a heterogeneous transition state ensemble with implications for the mechanism and regulation of mitochondrial membrane fusion.