Key result
Time-resolved tmFRET reveals GDP and Pi drive an unexpected Mfn1 conformational reversal in one cycle.
Why the study?
Allosteric mechanisms of nucleotide-driven self-assembly and conformational changes required for regulated mitochondrial membrane fusion by Mfn1 remain enigmatic due to incomplete structural information.
Time-resolved tmFRET reveals that the transition state of Mfn1 involves an equilibrium between open and closed states, redefining the understanding of GTP-driven conformational dynamics in mitochondrial membrane fusion.
Challenges models of Mfn1 GTPase cycling; leaves open relevance to cardiac mitochondrial fusion.
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 either 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. eTOC summary Hurwitz et al. leverage a novel FRET-based approach to quantify conformational dynamics of Mfn1, a member of the dynamin superfamily that mediates mitochondrial outer membrane fusion. Their findings shed light on how GTP hydrolysis governs the conformational state of Mfn1, as well as the energetics underlying a key conformational transition.
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Hurwitz et al. (2025) studied this question. GTP-coupled conformational dynamics of Mfn1 was evaluated on Conformational states and distance distributions. Time-resolved tmFRET revealed that the presence of GDP + Pi results in an equilibrium between open and closed states of Mfn1, demonstrating an unexpected conformational reversal in a single cycle.
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