The ability of dynamin to assemble into helices and constrict the underlying lipid bilayer is essential for vesiculation events in the cell. This study provides new insight into the mechanism of membrane vesiculation by examining conformational changes apparent in ΔPRD dynamin upon GTP addition. As observed previously, when comparing the non-constricted and constricted helical structures, the decrease in the number of subunits per turn (14.2 to 13.2, giving a ratio of 1.07) does not account for the change in circumference (a ratio of 1.25, 50 nm * π divided by 40 nm * π). If dynamin acted as a simple sliding ratchet upon constriction, 11.3 (14.2 divided by 1.25) subunits would be found in one turn of the constricted ΔPRD dynamin helix. Therefore, conformational changes within and between repeating subunits in the helical arrays of ΔPRD dynamin occur during constriction.
The computational fittings described here confirm the initial prediction placing the GTPase domain in the outer radial density, which is also confirmed by immunogold labeling, and the PH domain in the inner radial density. Moreover, the current fittings determine the packing within the repeating dimer subunit and the relative orientations of multiple dimers within the helical array. The remaining two domains, middle and GED, are positioned in the middle radial density, which undergoes the most dramatic conformational change upon helical constriction (red line in , and ). Here, we show that upon assembly, a region of the middle domain of dynamin is protected from proteolysis due to interactions in the helical array. This finding confirms the tight packing proposed for the middle domain and GED in the helical structure when lipid is present. The site of protection is in an area that recently has been shown to be essential for dynamin tetramer formation in solution (Ramachandran et al., 2006
). Protection of the middle domain from cleavage in the dynamin lipid tube suggests that a conformational rearrangement occurs upon lipid binding and/or assembly of the helical array.
A corkscrew model for dynamin constriction
The self-assembly of dynamin results in enhanced stability within or between adjacent GTPase domains, which may lead to more efficient hydrolysis within higher-ordered structures. In support of this hypothesis, the switch 2 region of the dynamin GTPase is adjacent to interface #1 (). Near this region, the shibire
ts2 mutation (G146, orange sphere) results in a defect in GTP binding at non-permissive temperatures (Narayanan et al., 2005
). Furthermore, mutations of another residue (T141) near the switch 2 region have been shown to enhance (T141A) or inhibit (T141D) assembly-stimulated GTPase activity (Song et al., 2004a
). Separately, a neighboring mutant (K142A) inhibits endocytosis despite having no affect on GTPase activity, and therefore may uncouple GTP hydrolysis from dynamin’s conformational change (Marks et al., 2001
). We pursued the possibility that the GTPase domain alone may form weak oligomers (see Experimental Procedures
), however, no oligomerization was found unless a GED fragment was present (data not shown), which is consistent with previous studies (Muhlberg et al., 1997
). Therefore, any interactions between adjacent GTPase monomers are dependent upon assembly promoted by other regions of the dynamin sequence, specifically the middle domain and GED.
After constriction, adjacent GTPase dimers are in a position to interact. The sequence in Interface #2 () is specific to dynamin, and several residues adopt different conformations when comparing the rat and Dictyostelium
structures. The peripheral sequence (purple loop in ) in the rat GTPase domain (Reubold et al., 2005
) is largely unstructured, while an additional α-helix is observed for the same region in the Dictyostelium
structures (Niemann et al., 2001
). The crystallographic B-factors for this region are higher in both structures indicating potential flexibility. This flexibility may be stabilized by self-assembly of the loop region, while elasticity in this region may be important for constriction to occur upon GTP addition.
Immediately downstream from the shibire ts1
mutation, GTPase-GED interactions have been proposed at the hydrophobic cleft between the N- and C-terminal helices of the GTPase domain (Niemann et al., 2001
). Changes in the structure and orientation of the GTPase domain, due to GTP binding (and later hydrolysis), are likely propagated to the GED/middle domain leading to the kinked structure observed for the middle density in the constricted state. The observed kinked pattern of middle radial density coincides with the twisting of the GTPase domains relative to one another, which allows for tighter packing of the GTPase domains in the constricted state. Overall, the combined GTPase/middle/GED subunit motions act like a corkscrew normal to the helical axis (). This motion along the helical array changes the subunit packing leading to compaction of the structures parallel to the helical axis and constriction normal to the lipid bilayer.
Comparing the PH domain fittings in the non-constricted and constricted maps also reveals a shift relative to the helical axis upon constriction (). However, adjacent PH domains are not in a position to interact or stabilize the oligomeric state through intermolecular interactions between adjacent dynamin monomers. Rather, the PH domain provides an affinity for lipid membranes with a negative potential, which concentrates and anchors dynamin to the membrane. As a result, the lipid bilayer provides a backbone for dynamin to assemble and undergo conformational changes upon nucleotide binding/hydrolysis. Without this backbone, dynamin spirals formed in vitro immediately disassemble when GTP is added. Most members of the dynamin family do not contain a PH domain, so the function of the PH domain is specific for its role in vesicle fission.
The relative intensities for the inner radial densities are different between the non-constricted and constricted maps of ΔPRD dynamin tubes. The dynamin PH domain (inner radius) and lipid bilayer densities are stronger, and therefore more uniform, in the non-constricted map. Constriction of ΔPRD dynamin due to nucleotide binding may weaken stable interactions between the dynamin PH domain and the lipid bilayer, and subsequent GTP hydrolysis results in release from the membrane. Accordingly, over time dynamin falls off the lipid bilayer in the presence of GTP (Danino et al., 2004
Based on the fittings of dynamin domains to the cryo-EM reconstructions, we propose that the GTPase, middle and GED domains work in concert to drive helical constriction. The PH domain acts as a separate entity during vesicle fission, tethering dynamin to lipid. Concurrent with dynamin recruitment to regions of negatively charged lipid, the middle domain and GED drive self-assembly of dynamin. In a cooperative manner, the GTPase domain is then stimulated, and upon nucleotide binding, undergoes a conformational change that is sensed and propagated through the middle/GED interaction. Overall, the conformational change resembles a corkscrew motion that acts to constrict the membrane through a combination of sliding (going from 14.2 to 13.2 subunits per turn) and twisting of each repeating subunit in the array (). Interactions between GTPase, middle and GED domains are more tightly packed, while PH domain interactions with the membrane may be placed under stress due to changes in lipid substrate curvature. Ultimately, fission relieves the strain placed on the membrane, and dynamin is released.
The reconstruction of ΔPRD dynamin in the constricted state represents a transition state between non-constricted and supercoiled states observed with both wild type and ΔPRD tubes upon GTP hydrolysis (Danino et al., 2004
; Roux et al., 2006
). In vivo
, if the dynamin helix were anchored at two positions (plasma membrane and coated pit), a coiled strain would be placed on the membrane possibly causing fission, which is consistent with the observed fragmentation of dynamin tubes due to GTP-induced twisting (Roux et al., 2006
; Sweitzer and Hinshaw, 1998
). Dynamin’s PRD-binding partners, such as amphiphysin (Grabs et al., 1997
), intersectin (Evergren et al., 2007
) and endophilin (Gad et al., 2000
; Ringstad et al., 1999
), also play a role in dynamin-dependent endocytosis. Amphiphysin has been shown to target dynamin to clathrin-coated pits (Shupliakov et al., 1997
) and increases dynamin’s rate of GTP hydrolysis and conformational change (Takei et al., 1999
). SH3-containing cofactors may bind the PRD of dynamin at late stages of endocytosis and prevent self-association of the PRD with the rest of the dynamin molecule, thereby allowing constriction to proceed. Therefore, the role of the PRD is limited to targeting and regulating dynamin constriction, while the GTPase, middle and GED domains, which are conserved throughout the dynamin family, comprise the mechanochemical core of dynamin that drives membrane fission. Overall, this work addresses how the conformational changes observed for dynamin in vitro
are essential for constriction during endocytosis in vivo
. Using a multifaceted approach, we are able to present the first model for the organization and conformational changes of the five distinct domains of dynamin during membrane fission.