Structural basis for mechanochemical role of Arabidopsis thaliana dynamin-related protein in membrane fission

Dear Editor, Dynamins and dynamin-related proteins (DRPs) constitute a large superfamily of GTPases throughout animal, plant, and bacteria and play essential roles in core cellular processes (Praefcke and McMahon, 2004). Plant specific dynamin-related subfamilies share essential functions with those in mammalian cell, e.g. clarthrinmediated endocytosis and fission of mitochondria; yet they also play unique functional roles in plant cells (Hong et al., 2003; Chen et al., 2011; Xue et al., 2011) (Supplementary Figure S1). Key features of dynamin members, including large molecular size, high basal GTP hydrolysis, and self-assembly into filamentous helices, distinguish them from other classical signaling and regulatory GTPases (Praefcke and McMahon, 2004). Dynamins are known to play a dual-role in clathrinmediated endocytosis, in which the basal activity is necessary for early endocytic events and an assembly-stimulated activity is required in later stages of membrane fission (Sever et al., 1999). A mechanochemical model was presented focusing on dynamin in triggering the vesicle scission stimulated by GTP hydrolysis. In such a model, two distinct mechanisms, i.e. ‘pinchase’ and ‘poppase’, were proposed based on GTP hydrolysis induced dynamin vesiculation on liposomes (Sweitzer and Hinshaw, 1998). Differences between the two possible mechanisms focus on tightening the vesicle neck by dynamin oligomer to ‘pinching off’ the vesicle or a length-wise extension of the dynamin super helix to ‘popping off’’ of the vesicle mechanochemically (Praefcke and McMahon, 2004). To clarify the assembling process and working mechanism of dynamin members in plant cell cytokinetic processes, we initiate the functional and structural investigation on Arabidopsis thaliana dynaminrelated protein 1A (AtDRP1A). GED of dynamin members is known to be directly associated with the GTPase domain (Chappie et al., 2009) and plays a crucial role in either assembly-stimulated or basal GTPase activity. We therefore engineered a 40-kDa AtDRP1A variant containing the GTPase domain and C-terminal segment of GED (CGED, residues 585–606) fused by a flexible linker (Figure 1A) (named as AtDRP1A GG hereafter) as suggested in human dynamin (Chappie et al., 2009). The purified AtDRP1A GG protein maintained a GTPase activity with Km value of 563 mM and kcat of 0.11 min 21 (Supplementary Figure S2), which is in the range of the dynamin family (Praefcke and McMahon, 2004) and suggests that this variant represents the basic catalytic machinery of AtDRP1A. Substrate-dependent oligomerization is known to play a central regulatory role in a number of G proteins (Gasper et al., 2009) including the activity of dynamin’s GTPase domain (Chappie et al., 2010). AtDRP1A GG existed in a monomeric form in the presence of either GDP alone or nonhydrolysable GTP analogues as well as in the absence of additional nucleotides (Supplementary Figure S3A), while mainly existed in a dimeric form when it was incubated with GDP supplemented with NaF and AlCl3 (Supplementary Figure S3A). A hexagonal and a monoclinic crystal form were subsequently obtained with AtDRP1A GG both in dimeric state (Figure 1B). In both crystal forms, GTPase domain presented a canonical structure of GTPase family and two adjacent GTPase cores associated symmetrically with each other (Supplementary Figure S3B). Among the interacting residues, NQDLATSD AIK, named as the ‘trans stabilizing loop’ (Chappie et al., 2010), played a major role in AtDRP1A GG dimerization (Supplementary Figure S3C). Particularly, D186 interacted with G65 thus participating in P-loop stabilization. The side chain of D217 and K218 in the conserved G4 (TKID) motif contributed to intradimer interaction through contacting with the residues in a ‘dynamin specific loop’ (Chappie et al., 2010). More importantly, the hydrophilic side chain of D217 formed in trans interactions with bound GDP molecule as well as G4 motif of the symmetry mate (Supplementary Figure S3D). Although D186A and D217A mutants both forfeit the substrate-dependent dimer formation (Supplementary Figure S4), D186A showed no detectable influence on basal GTPase activity, while D217A significantly reduced GTPase activity (Figure 1C). These observations were consistent with the structural result that D186 is merely involved in protein–protein interaction while D217 also participated in stabilizing GTP molecule. These results reveal that, like in mammalian dynamins (Chappie et al., 2010), oligomerization of AtDRP1A is essential for its stimulated GTP hydrolysis but not the basal GTPase activity. Significantly, distinct differences on ligand binding, active site conformations, and orientations of bundle signaling element (BSE) were observed between the two crystal forms. Whereas the GDP molecule was identified in both crystal forms, AlF4 , resembling the g-phosphate group in the GTP hydrolysis transition status, Mg2+ and Na+, which are crucial for GTP hydrolysis, were only observed in the monoclinic crystal form (Figure 1D). As a consequence of 3.6 Å back bond 378 | Journal of Molecular Cell Biology (2011), 3, 378–381 doi:10.1093/jmcb/mjr032