Structural basis for GTP hydrolysis and conformational change of MFN1 in mediating membrane fusion

Abstract

Fusion of the outer mitochondrial membrane is mediated by the dynamin-like GTPase mitofusin (MFN). Here, we determined the structure of the minimal GTPase domain (MGD) of human MFN1 in complex with GDP-BeF. The MGD folds into a canonical GTPase fold with an associating four-helix bundle, HB1, and forms a dimer. A potassium ion in the catalytic core engages GDP and BeF (GDP-BeF). Enzymatic analysis has confirmed that efficient GTP hydrolysis by MFN1 requires potassium. Compared to previously reported MGD structures, the HB1 structure undergoes a major conformational change relative to the GTPase domains, as they move from pointing in opposite directions to point in the same direction, suggesting that a swing of the four-helix bundle can pull tethered membranes closer to achieve fusion. The proposed model is supported by results from in vitro biochemical assays and mitochondria morphology rescue assays in MFN1-deleted cells. These findings offer an explanation for how Charcot-Marie-Tooth neuropathy type 2 A (CMT2A)-causing mutations compromise MFN-mediated fusion.

Metabolic implications of organelle–mitochondria communication

“…It is still not clear how many MFN1, MFN2, and OPA1 molecules oligomerize to carry out this function. Dimeric and tetrameric interaction models have been proposed . As mentioned before, MFN2 is present at both sides of the MAM, and it participates in mitochondria–ER tethering complexes .…”

The structural biology of the dynamin‐related proteins: New insights into a diverse, multitalented family

“…The successful GG design was duplicated to examine the catalytic regions of other dynamin relatives, including Arabidopsis thaliana DRP1A, Drp1 (named DNM1L in the study) (Box ), MxA and Vps1 . Similar approaches have also been successful in the structural characterization of the outer mitochondrial membrane fusion DRP Mitofusin 1 …”

“…The HB1 bundles extend out linearly from the G domains—oriented perpendicular to the 2‐fold symmetry axis—such that the dimer measures 180 å in length. HB1 rotates downward by 82° in the structure of mini‐Mfn1 complexed with GDP.BeF 3, mirroring the nucleotide‐dependent conformational rearrangements observed in other DRPs. Thus, while Mfn1 does not have a true distinct BSE, HB1 may be functionally analogous.…”

“…Rotation around a second hinge connecting Neck to the G domain (Hinge 2) plays a critical role in this transition. Interestingly, the resulting Neck conformation mirrors that seen in the structure of the GDP.BeF 3 ‐bound mini‐Mfn1 . These similarities suggest BDLP and Mfns define a distinct subclass that likely shares the same molecular mechanism and comparable functions.…”

“…13 Similar approaches have also been successful in the structural characterization of the outer mitochondrial membrane fusion DRP Mitofusin 1. [14][15][16] The first structural characterization of the "Stalk" of a dynamincomprising the helical segments of the Middle Domain and GED-was unexpectedly obtained during attempts to crystallize full-length MxA ( Figure 1D). 20 The recombinant protein suffered unintended proteolysis at some point during purification and/or the crystallization process.…”