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

Area of Science:

• Cell biology
• Molecular biology
• Biophysics

Background:

• Outer mitochondrial membrane fusion is essential for cellular function and relies on mitofusins (Mfn1 and Mfn2).
• The nucleotide-driven conformational changes of mitofusins are critical for membrane fusion, but the precise allosteric mechanisms remain unclear due to limited structural data.

Purpose of the Study:

• To investigate the GTP-coupled conformational dynamics of mitofusin 1 (Mfn1) using time-resolved transition metal ion fluorescence resonance energy transfer (tmFRET).
• To elucidate the structural mechanisms underlying Mfn1's role in mitochondrial membrane fusion.

Main Methods:

• Utilized a minimal Mfn1 construct (GTPase domain and helical bundle 1 connected by Hinge 2).
• Engineered Förster Resonance Energy Transfer (FRET) pairs using fluorescent noncanonical amino acids and metal ion acceptors.
• Measured tmFRET and fluorescence lifetimes across the catalytic cycle to determine distance distributions and capture structural heterogeneity.

Main Results:

• Confirmed an open conformation for the GDP-bound state of Mfn1 in solution.
• Revealed that the transition state is not a single closed conformation but an equilibrium between open and closed states when GDP + Pi is present.
• Identified that GTP binding favors the open state, and the apo state exhibits a distinct conformation.

Conclusions:

• GTP-driven conformational dynamics in Mfn1 involve an unexpected conformational reversal within a single catalytic cycle.
• The transition state is a heterogeneous ensemble, not a single structure, impacting the understanding of Mfn1 regulation.
• These findings provide new insights into the mechanism and regulation of mitochondrial membrane fusion.