A membrane-inserted structural model of the yeast mitofusin Fzo1

Abstract

Mitofusins are large transmembrane GTPases of the dynamin-related protein family, and are required for the tethering and fusion of mitochondrial outer membranes. Their full-length structures remain unknown, which is a limiting factor in the study of outer membrane fusion. We investigated the structure and dynamics of the yeast mitofusin Fzo1 through a hybrid computational and experimental approach, combining molecular modelling and all-atom molecular dynamics simulations in a lipid bilayer with site-directed mutagenesis and in vivo functional assays. The predicted architecture of Fzo1 improves upon the current domain annotation, with a precise description of the helical spans linked by flexible hinges, which are likely of functional significance. In vivo site-directed mutagenesis validates salient aspects of this model, notably, the long-distance contacts and residues participating in hinges. GDP is predicted to interact with Fzo1 through the G1 and G4 motifs of the GTPase domain. The model reveals structural determinants critical for protein function, including regions that may be involved in GTPase domain-dependent rearrangements.

Structural dataset from microsecond-long simulations of yeast mitofusin Fzo1 in the context of membrane docking

“…The structural models presented here build upon an experimentally validated model of the monomeric unit, namely our previously published model of Fzo1 in a closed conformation [2]. It is first used to generate a monomeric model of the open conformation.…”

“…Two chains of the Fzo1 model in closed conformation [2] were placed by superimposing their GTPase domains onto that of BDLP in open conformation [8]. Then, only the coordinates of the two fragments that comprise the Fzo1 GTPase domain (residues 188–461) were retained to form the final GTPase domain dimer model.…”

“…Two Fzo1 chains in closed conformation [2] were oriented facing each other within a compatible distance to accommodate two interacting GTPase domains. Subsequently, the coordinates of residues 188–440 enclosed between hinges 2a and 2b (i.e., comprising the GTPase domain) were removed from both chains and replaced with the GTPase dimer construct described above.…”

“…Two chains of the Fzo1 model in closed conformation [2] were manually oriented with respect to each other to generate the back-to-back interaction. In the resulting model system, the HR domains face each other in a parallel fashion.…”

“…Starting from our previous Fzo1-BDLP target-template alignment [2], we introduced homologous chain breaks on the Fzo1 model [2], resulting in five rigid blocks. Each fragment was superposed to its corresponding fragment in 2W6D to reconstitute the orientation found in BDLP.…”