Design and Characterization of a Minimal GTPase-GED Fusion
The structures of the dynamin A and rat dynamin GTPase domains (Niemann et al., 2001
; Reubold et al., 2005
) each revealed that a helix derived from the myosin fusion protein associates with a hydrophobic groove on the GTPase domain formed by its N- and C-termini (NGTPase
; A). This interaction was suggested to mimic GED docking, but which region of GED might dock at this site had not been identified. The discovery that second site mutations located at the C-terminus of GED rescue the phenotype of a GTPase domain mutant of dynamin (Narayanan et al., 2005
) attracted our attention to this portion of GED.
Figure 1. Model of dynamin GG interface. (A) Crystal structure of rat dynamin GTPase domain (green) fused to myosin motor core (PDB: 2aka). A helix from the myosin (red) packs onto a hydrophobic groove comprised of the N- and C-termini (teal) of the GTPase domain. (more ...)
Secondary structure prediction algorithms model the C-terminus of GED (CGED
) as a continuous α-helix (Chugh et al., 2006
). A sequence alignment of dynamin family members revealed a number of highly conserved hydrophobic residues that map to one side of this putative CGED
helix. (, B and C). Given the similar amphipathic nature of the helices that constitute the predicted GED docking site, we speculated that the NGTPase
, and CGED
form a three-helix bundle (C). We set out to test this hypothesis by designing a minimum GTPase-GED construct (, A and B).
Figure 2. Design and functional characterization of a minimal GTPase-GED fusion (GG). (A) Design of GG fusion. Dashed line represents flexible linker connecting the GTPase and GED fragments. (B) Diagram depicting the putative folding of GG. The crystal structure (more ...)
Dynamin's GTPase domain is insoluble when expressed in E. coli as an MBP fusion (C). This instability could reflect exposure of the hydrophobic groove between NGTPase and CGTPase to solvent in the absence of the GED or myosin helix. Efforts to recover the GTPase domain by coexpression with GED failed (J. S. Chappie, unpublished data), suggesting that protein folding initially drives this interaction. If our hypothesis was correct, we reasoned that we could instead stabilize the GTPase domain by tethering the C-terminal portion of GED with a short flexible linker (A). This design assumes that the incorporated GED fragment will form a helical peptide and dock with NGTPase and CGTPase in a manner analogous to the myosin helix (B). As we predicted, expression of this GG fusion rescues the protein to the soluble fraction (C), even after removal of the MBP fusion tag (D). Purification yields a 39.3-kDa protein that is capable of hydrolyzing GTP at a rate comparable to the basal GTPase activity of full-length dynamin (E). This activity can be inhibited by a P-loop mutation known to disrupt GTP binding in dynamin (S45N, E). GG exists as a monomer in solution (data not shown), and we did not observe any detectable assembly at high concentrations of protein (>250 μM) or at low salt. Moreover, the observed hydrolysis activity is independent of protein concentration (data not shown). These data establish that the C-terminus of GED can provide structural stability through its interactions with the GTPase domain, allowing the latter to perform its basic chemical function outside the framework of the full-length protein. Monomeric GG and the full-length dynamin tetramer exhibit comparable GTPase activities, which strongly argues that the four GTPase domains within the tetramer function independently to hydrolyze GTP in the unassembled state. Although outside the scope of these studies, the minimal GG construct described here should also be a useful tool for future structure-function studies aimed at defining the mechanism of dynamin's basal GTPase activity.
Chemical Cross-Linking Confirms Direct Interaction of CGED with NGTPase
GG's activity implies that our design reconstitutes the minimal structural interactions required for dynamin's basal GTPase activity. However, this does not provide direct structural evidence that the GED interacts with the GTPase domain. In the absence of a high-resolution structure, we utilized chemical cross-linking to define this interaction further. Thiol-specific cross-linkers have been used extensively as molecular rulers to determine inter- and intramolecular distances, thereby providing constraints to guide structural modeling (Kenyon and Bruice, 1977
; Loo and Clarke, 2001
; Dalmas et al., 2005
). In these cases, a bifunctional cross-linker is mixed with a protein containing two reactive cysteines, and cross-linked products are observed by nonreducing SDS-PAGE. The fixed position of the cysteines and the length of the cross-linker spacer arm each impose a distance constraint that determines the success of the cross-linking reaction. By varying the cross-linker length, the distance between the sulfhydryl side chains can be estimated. The extensive hydrophobic interface in the putative three-helix bundle would restrict the possible orientations of the amphipathic CGED
helix in GG, making it an ideal template for this type of cross-link mapping.
To facilitate cross-linking, we generated a series of double cysteine mutants in GG and reacted them with a panel of bifunctional MTS compounds that ranged in length from 3.6 to 7.8 Å (, A and B). Each cysteine pair is comprised of one substitution in the GTPase domain (R15C in the N-terminus or R297 in the C-terminus) and one substitution from an array of engineered cysteines in the GED (R730C, H733C, K736C, or S740C; A). An additional mutation that removed a surface accessible reactive cysteine (C86S) was introduced into all constructs to limit nonspecific and intermolecular cross-linking. A third, partially buried cysteine (C169), thought to be critical for GTPase activity (Ramachandran and Schmid, 2008
), was left unchanged. We directed our mutagenesis to the hydrophilic surfaces of the respective helices (C) so as not to interfere with their hydrophobic packing and confirmed that the cysteine mutagenesis did not alter GG's GTPase activity (data not shown).
Figure 3. Intramolecular cross-linking of GTPase and GED helices. (A) Residues selected for double cysteine mutagenesis in GG. Cysteine pairs included one substitution in the GTPase domain (R15C in NGTPase or R297C in CGTPase) and one substitution in the CGED region (more ...)
The cross-linking profile for each sample was visualized using nonreducing SDS-PAGE and Coomassie staining (C). We detected efficient cross-linking between R15C in NGTPase and both R730C and H733C in CGED, as evidenced by a gel shift to a faster migrating species (C, left panels). Mass spectrometry analysis of treated samples confirmed that the small gel-shift seen with the R15C/R730C construct corresponds to the presence of MTS-mediated covalent interactions between the engineered cysteines located in the NGTPase and CGED (Supplementary Figure S1). Although the efficiency increased with increasing length of the cross-linking reagent, we detected significant cross-linking even in the presence of the shortest reagent MTS-1–MTS, which approximates a distance of 3.6 Å between the cysteine pair. These findings confirm a close association of the NGTPase and CGED helix. We also detected slight gel shifts in the R15/K736C and R15C/S740C constructs, but only in the presence of longer cross-linking agents. However, the smaller degree of this gel shift makes interpretation more difficult. Overall, these data agree with our model for GED docking to the N-terminal helix of the GTPase domain.
In contrast, we were unable to detect cross-linking in the R297C GG mutants by SDS-PAGE. (C, compare right panels with C86S control). This discrepancy may reflect an inherent difference in SDS-PAGE mobility for the two intramolecularly cross-linked species formed in GG. A covalent interaction between the NGTPase and CGED (R15C double mutants) connects the two termini of the GG construct, dramatically altering the shape of the unfolded protein; cross-linking between the CGTPase and CGED only modifies the structure of the extreme C-terminus, leaving the majority of the construct undisturbed. Thus, the shift in migration produced by the latter change may be so minor that it is not resolved by SDS-PAGE. Mass spectrometry analysis of the CGTPase-CGED cross-linked samples also proved ambiguous, as the corresponding CGTPase mutant peptide could not be detected, even in the non–cross-linked control digests. Thus, we cannot draw conclusions from these negative findings as to the positional relationship between CGTPase and CGED. However, the mutagenesis studies described below support an interaction between these two helices.
Perturbation of the GG Interface Disrupts Dynamin GTP Hydrolysis
To probe the functional significance of the GTPase-GED interface, we next generated a series of point mutations targeting the highly conserved hydrophobic residues within each of the three interface helices (B). Mutation of a number of these side chains to alanine in GG, both individually and in pairs, produced no effect on GTPase activity (data not shown). Although it is possible that the alanine substitutions were maintaining rather than disrupting the conserved hydrophobic interface, we suspected that these interactions might be specifically involved in modulating dynamin's assembly-stimulated activity. Therefore, for subsequent analyses we decided to engineer mutations in the context of full-length dynamin. Initial mutagenesis of several hydrophobic residues in GED to alanine also yielded proteins whose activities were indistinguishable from wild type (data not shown). Therefore, we introduced asparagine residues and determined how each substitution affected basal and assembly-stimulated hydrolysis.
Preliminary screening experiments identified mutations in each interface helix that differentially affect dynamin GTPase activity (), thereby providing strong evidence that our putative three-helix bundle at the GTPase-GED interface is functionally relevant. Comparison of the normalized rates to wild-type dynamin reveals two classes of interface mutants. The first class (I10N, L293N, L296N, and L300N) is severely defective in stimulated GTPase activity, with some mutants such as L293N and L300N also exhibiting a basal defect. The second class (L12N, F20N, and A738N) is partially defective in stimulated turnover, with phenotypes ranging from 40 to 60% of the normal activation. Interestingly, A738 corresponds to the residue mutated in a Sushi
allele (A738T) that was shown to rescue function of a dynamin GTPase domain mutant in vivo (Narayanan et al., 2005
). The small increase in basal GTPase activities observed for some mutants in this preliminary screen were not reproducible and may reflect small amounts of aggregated species present in individual preps.
Figure 4. Basal and assembly-stimulated GTPase activities of interface mutants in vitro. Normalized activity for each mutant is expressed as a percentage of wild-type activity. All mutations were assayed in the context of full-length dynamin at a concentration (more ...)
Class I Interface Mutations Destabilize Dynamin Structure
Because the GTPase-GED interface is primarily hydrophobic in nature, it is possible that mutations in this region could have significant effects on the structural integrity and proper folding of dynamin. We analyzed the interface mutants by sedimentation and EM to determine if the observed phenotypes were related to structural irregularities (). In the absence of liposomes, wild-type dynamin exists as tetramers in solution that remain in the soluble fraction after sedimentation (A). Addition of PIP2-containing liposomes shifts the protein to the pellet due to self-assembly on the charged membrane template (B). Under these conditions, wild-type dynamin forms large, decorated tubes that are visible by negative-stain EM (C). Analysis of the interface mutants shows that the class I mutations pellet even in the absence of liposomes (A). When visualized by EM, these proteins form large, amorphous aggregates on the grid (data not shown). Only L296N retains the capacity for self-assembly and tubulation, which likely reflects the small amount of the protein that is still soluble and unassembled in the absence of liposomes (, A and B). The length and morphology of these L296N decorated tubes differ significantly from wild-type dynamin, as evidenced by the incomplete pattern of decoration. On the basis of these observations, we conclude that the class I interface mutants (I10N, L293N, L296N, and L300N) destabilize dynamin structure and that the observed defects in stimulated GTPase activity result from aggregation of the protein and the inability to self-assemble properly. These structural defects could also explain the basal hydrolysis defects observed in L293N and L300N.
Figure 5. Self-assembly properties of interface mutants. (A) Sedimentation of interface mutants (1 μM) in the absence of liposomes. After sedimentation, supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE. T, total amount of protein in the (more ...)
Class II Interface Mutations Uncouple Stimulated GTPase Activity from Dynamin Assembly
In contrast to the class I mutants, the class II mutants (L12N, F20N, and A738N) remain in the soluble fraction when subjected to centrifugation in the absence of liposomes (A). When incubated with PIP2-containing liposomes, these proteins sediment and assemble normally, forming decorated tubes that are indistinguishable from wild-type dynamin (, B and C). At this low resolution, the packing along these tubes is apparently unaltered by changes in the GTPase-GED interface, indicating that these proteins are competent for self-assembly. The specificity of these biochemical phenotypes implies that the structural integrity of these proteins is preserved and that these mutations uncouple stimulated GTPase activity from dynamin assembly.
To investigate this further, we determined the kinetic parameters (kcat and Km) of the basal and assembly stimulated GTPase activities of the class II interface mutants. These studies were performed on several independently purified batches of WT and mutant dynamin to confirm the reproducibility of our findings. Although all three mutants exhibit robust basal rates of GTP hydrolysis (), the L12N and A738N mutants are reduced relative to WT. Importantly, the Km for basal hydrolysis, which is a close approximation of GTP-binding affinity (), is unaffected, further confirming that the GTPase domains of these mutants are properly folded. The stimulated GTPase activity was diminished in each mutant (), although we observe significant batch-to-batch variation under these conditions. Though the measured Km values for stimulated activity are a less accurate representation of binding affinity because of variable and more rapid rates of GTP hydrolysis, the mutants still do not appear to disrupt GTP binding relative to WT. Together these data suggest that the GG interface plays a more selective role in modulating assembly-stimulated GTPase activity in addition to its critical function in stabilizing dynamin structure.
Michaelis-Menten kinetic parameters for basal and assembly- stimulated GTPase activities of wildtype dynamin and GTPase-GED interface mutants
Class II Interface Mutants Reduce Dynamin-Catalyzed Membrane Fission In Vitro
Dynamin's role in CME depends on its ability to catalyze membrane fission, which has recently been shown to require localized curvature imposed by the assembly of GTPase-limited, short dynamin collars (Pucadyil and Schmid, 2008
; Bashkirov et al., 2008
). Longer dynamin assemblies that can only assemble in the absence of GTP were shown to stabilize underlying tubular membranes and were unable to mediate membrane fission. Thus, dynamin-catalyzed fission critically depends on GTPase-regulated cycles of dynamin assembly and disassembly (Ramachandran and Schmid, 2008
). In this context, we anticipated that the assembly-stimulated defects exhibited by class II mutants would translate into a reduction in membrane fission by dynamin. To test this prediction, we used a recently developed assay that measures vesicle formation from supported bilayers with excess membrane reservoir (SUPER templates; Pucadyil and Schmid, 2008
) and examined the ability of the L12N, F20N, and A738N mutants to catalyze membrane fission. SUPER templates containing fluorescent lipid were mixed with dynamin in the presence or absence of GTP and then centrifuged at low speed. Fission was detected as an increase in the fluorescence intensity of the supernatant resulting from the liberation of lipid vesicles (). As can be seen, L12N is significantly impaired in its ability to catalyze membrane fission and vesicle release, whereas A738N only shows a partial defect and F20N appears to function as effectively as WT. Unexpectedly, the degree to which each mutant can facilitate fission in vitro does not fully correlate with the severity of its assembly-stimulated defect, suggesting that other aspects of dynamin function not reflected in its in vitro GTPase activities but differentially dependent on the GTPase-GED interface are important for dynamin-catalyzed fission.
Figure 6. Membrane fission by class II interface mutants in vitro. Fluorescent SUPER templates were prepared as described in Materials and Methods. ■, fission by 0.5 μM dynamin in the absence of nucleotide; □, fission in the presence of (more ...)
Class II Interface Mutants Affect the Late Stages of Endocytosis In Vivo
Because L12N reduced stimulated activity and fission in vitro, we wanted to know if this change at the GTPase-GED interface also adversely affects CME in vivo. To examine this possibility, we performed experiments in Dyn2 KO cells reconstituted with GFP-tagged WT, L12N, F20N, or A738N Dyn2 proteins. Stable cell lines expressing these mutants at levels equivalent to endogenous Dyn2 were generated, and then endogenous Dyn2 was excised by introduction of Cre recombinase (Liu et al., 2008
). We chose this system to avoid the potential of stronger and/or nonspecific defects caused by overexpression of these mutants in the presence of endogenous Dyn2. The uptake of biotinylated-Tfn (BSS-Tfn) was determined either by inaccessibility to avidin, a large bulky probe, or to MesNa, a small, membrane-impermeant reducing agent. Sequestration of BSS-Tfn from avidin assesses the formation of both constricted coated pits and coated vesicles, whereas MesNa resistance is only acquired after BSS-Tfn has been internalized into sealed coated vesicles (A). We were unable to detect an in vivo defect in Dyn2 KO cells reconstituted with the A738N mutant. Although not correlating with impaired GTPase and fission activities of this mutant, this finding was consistent with the fact that mutation of this residue can restore function of the GTP-binding defective, shits2
dynamin mutant. In contrast, the in vivo phenotype of the L12N mutation correlated well with its in vitro properties. L12N partially blocked uptake into both the avidin- and MesNa-inaccessible compartments; however, there was a greater degree of inhibition when assessed by MesNa resistance than avidin inaccessibility (, B and C). These data indicate that L12N partially impairs the later stages of endocytosis, leading to an accumulation of BSS-Tfn in constricted coated pits that are inaccessible to avidin but remain accessible to MesNa as would be predicted from its in vitro defects. We also detect a slight inhibition of uptake for F20N in the MesNa assay (C), although not in the avidin assay (B). These phenotypes are consistent with a late effect on CME, resulting from reduction of either the rate or efficiency of membrane fission.
Figure 7. Class II interface mutants disrupt clathrin-mediated endocytosis in vivo. (A) Diagram of endocytosis intermediates and their accessibility to the avidin and MesNa probes. (B and C) Dynamin-2 knockout (Dyn2 KO) cells expressing either GFP (○) or (more ...)
To gain further insight into the stage at which these mutations impaired CME, we inspected the clathrin-coated pit (CCP) intermediates in our reconstituted Dyn2 KO cells using indirect immunofluorescence (). Permeabilized cells expressing GFP-tagged Dyn2 constructs (WT, L12N, F20N, and A738N) were fixed and stained with the anti-ΑP2 antibody AP6. We found there to be no detectable difference in the number of coated pits between the class II interface mutants and WT Dyn2; however, short, tubule-like formations with associated dynamin puncta and decorated with AP2 were present in cells reconstituted with L12N (, enlarged boxes). Although the resolution of these experiments precludes a detailed description of these structures, the pattern of staining indicates that AP2 can bind along the length of these tubules. The accumulation of tubular structures in L12N is consistent with a defect in fission and correlates with its stronger inhibition of Tfn uptake in the MesNa assay.
Figure 8. L12N alters the morphology of clathrin-coated pits in reconstituted Dyn2 KO cells. Clathrin-coated pits were visualized by indirect immunofluorescence using the AP6 mAb directed against the α-adaptins of AP2. Dynamin-2 constructs (WT, L12N, F20N, (more ...)
To further explore the possibility that these structures represent delayed fission events we examined the endocytic intermediates in these cells by high-resolution EM. Plastic-embedded thin sections of reconstituted Dyn2 KO cells were imaged and the CCP intermediates for each mutant were counted and scored according to their morphology as either shallow, invaginated, or constricted (). Invaginated pits are visibly connected to the membrane in a single thin section and thus are assumed to correspond to avidin-accessible structures (A). Given that uncoating is rapid and that endocytic vesicles rapidly translocate away from the cell surface, coated vesicle structures closely apposed to the plasma membrane are assumed to represent constricted intermediates, whose narrow necks are not captured in these individual thin sections (A). This classification has been used and validated extensively in past studies, under conditions in which CME is known to be inhibited (Schmid and Carter, 1990
; Sandvig et al., 1987
Figure 9. L12N increases the number of late endocytic intermediates in reconstituted Dyn2 KO cells. (A) Thin sections of Dyn2 KO cells reconstituted with GFP-tagged Dyn2 proteins (WT, L12N, and F20N and A738N, as indicated) were prepared and imaged as described (more ...)
Based on this quantification, we observe a statistically significant increase in the number of constricted CCPs in L12N versus WT (B). This increase in late intermediates is seen at the expense of the invaginated and shallow CCPs, suggesting that L12N specifically hinders the late stages of clathrin-mediated endocytosis by delaying dynamin-dependent fission. Such a delay would impede the formation of coated vesicles, consistent with the stronger inhibition of Tfn uptake by L12N as detected by MesNa resistance. F20N also showed a slight inhibition of endocytosis in the MesNa assay, but we did not detect any significant change in the distribution of CME intermediates for this mutant by our EM analysis (B). Similarly, we detect no increase in late CME intermediates in cells expressing A738N, which mirrors the WT behavior of this mutant in the Tfn uptake assays. Coated vesicle-like structures accumulated adjacent to the plasma membrane only in cells expressing the L12N mutant. That this mutant shows the greatest defect in our in vitro membrane fission assay and the strongest defect in vivo supports our assumption that these structures are indeed constricted CCPs and not detached CCVs. Together these data suggest that the GG interface is critical for coordinating dynamin activity at late stages of CCV formation.