Dyn1-R361S and Dyn1-R399A are defective in self-assembly
Dynamin self-assembles into higher-order structures in low ionic strength buffers (<50 mM salt) even in the absence of a lipid template, pointing to the involvement of charged residues in protein–protein interactions (Hinshaw and Schmid, 1995). Hence, we chose to substitute two conserved arginines at positions 361 and 399 in the N-terminal half of the middle domain with uncharged residues, serine (R361S) and alanine (R399A) respectively, with the expectation that these mutations would adversely affect higher-order self-assembly. The CD and Trp emission spectra of Dyn1-R361S and Dyn1-R399A were identical to those of Dyn1-WT (wild type) suggesting that the tertiary structure of dynamin was not significantly perturbed by these mutations (Supplementary Figure 1).
Formation of higher-order structures consisting of rings and spirals onto negatively charged lipid templates including L-α-phosphatidylinositol-4,5-bisphosphate (PI4,5P2)-containing liposomes enhances dynamin's GTP hydrolysis rate up to 100-fold, a lack of or reduction of which may imply an intrinsic defect in self-assembly (Song et al, 2004b). To test whether substitutions R361S and R399A affect dynamin's assembly-dependent GTPase stimulation, we determined the GTPase activities of Dyn1-R361S and Dyn1-R399A upon incubation with 15 mol% PI4,5P2-containing liposomes (Figure 1A). The stimulated GTPase activities of Dyn1-R361S and Dyn1-R399A were reduced 3- and 10-fold, respectively, when compared with Dyn1-WT (Table I), suggesting that these mutants may indeed be impaired in higher-order self-assembly. More drastic reductions in stimulated GTPase activities were observed for Dyn1-R361S and Dyn1-R399A upon incubation with 100 mol% phosphatidylserine (PS) liposomes, another negatively charged lipid template that supports dynamin self-assembly (Supplementary Figure 2).
Figure 1. Dyn1-R361S and Dyn1-R399A are defective in self-assembly. (A) GTPase activities of 0.5 μM Dyn1-WT (○), Dyn-R361S (▪) or Dyn1-R399A (⧫) after pre-incubation with PI4,5P2-containing liposomes (150 μM lipid) was measured at 37°C, as described under Materials and methods. The concentration of Pi released is plotted as a function of time. The average values of at least four independent experiments are shown with standard deviations, as indicated. (B) Self-assembly of dynamin (1 μM protein) on liposomes (300 μM lipid) with or without PI4,5P2, or at low ionic strength (no salt), was examined by sedimentation followed by SDS–PAGE analysis of the supernatant (S) and pellet (P) fractions as described under Materials and methods; total protein (T). (C) Electron micrographs of Dyn1-WT, Dyn1-R361S and Dyn1-R399A (1 mg/ml protein) self-assembled on PI4,5P2-containing liposomes (1 mg/ml lipid) were obtained as described under Materials and methods. Scale bar, 200 nm.
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Table 1. Properties of Dyn1-WT, Dyn1-R361S and Dyn1-R399A
|Assembly-dependent GTPase activity (μM/min)a||90.3±6.9||30.1±1.7||8.9±3.5|
|Sedimentation coefficient (S or Svedbergs)b||∼10–12||∼6||∼6|
|Molecular mass by MALLS (kDa)||∼350||∼170||∼180|
|Estimated number of subunitsc||4||2||2|
The apparent reduction in the assembly-stimulated GTPase activities of Dyn1-R361S and Dyn1-R399A can be accounted for either by a loss of catalytic activity but not self-assembly or by impaired self-assembly that ultimately affects GTPase activity. To directly test these possibilities, we next determined the ability of these mutants to bind to, and form higher-order structures on, PI4,5P2-containing liposomes that are readily sedimentable by centrifugation. Although nearly all of Dyn1-WT was recovered by sedimentation in the presence of these liposomes, only ∼50% of each of the mutants was sedimentable (Figure 1B), confirming that Dyn1-R361S and Dyn1-R399A are impaired in membrane binding. In control experiments with liposomes lacking PI4,5P2 or PS, neither Dyn1-WT nor the mutants were sedimentable, reflecting dynamin's specificity towards negatively charged membranes. Consistent with a defect also in self-assembly, both Dyn1-R361S and Dyn1-R399A were impaired in assembling into sedimentable, higher-order structures under low ionic strength conditions that otherwise promote Dyn1-WT assembly even in the absence of a lipid template (Figure 1B).
The nearly 50% reduction in Dyn1-R361S and Dyn1-R399A binding to PI4,5P2-containing liposomes is less severe than the ∼70 and 90% reductions in their assembly-stimulated GTPase activities, respectively (Figure 1A and B). One possible explanation for this apparent inconsistency is a defect in the higher-order structures formed by membrane-bound Dyn1-R361S and Dyn1-R399A that results in a less effective stimulation of GTP hydrolysis. To determine whether the higher-order structures formed by Dyn1-R361S and Dyn1-R399A on PI4,5P2-containing liposomes are similar to those formed by Dyn1-WT, we visualized them by negative-stain electron microscopy. In contrast to Dyn1-WT, which deformed spherical liposomes into lipid tubules of nearly uniform diameter encircled by well-organized stacks of a polymeric dynamin spiral, both Dyn1-R361S and Dyn1-R399A formed lipid tubules of variable diameter along the tubule length encircled by disordered rungs of a dynamin spiral (Figure 1C). Taken together, these data suggest that Dyn1-R361S and Dyn1-R399A are not only impaired in membrane binding, but also form defective higher-order structures when self-assembled on membranes.
Dyn1-R361S and Dyn1-R399A are assembly-defective dimers
Dynamin binds to PI4,5P2-containing membranes via its PH domain. Previous studies have shown that the affinity of an isolated dynamin PH domain for the phosphoinositide headgroup is low, and therefore, stable binding of dynamin to a PI4,5P2-containing membrane surface requires the high avidity interactions provided by its oligomerization (Klein et al, 1998; Lemmon and Ferguson, 2000). Sedimentation equilibrium experiments have shown that dynamin in the unassembled state exists in dynamic equilibrium between monomeric and tetrameric states, with the tetramer thermodynamically favored over the monomer (Binns et al, 1999). As dynamin in the tetrameric state is expected to exhibit a higher avidity for the target membrane than a monomer or a dimer, we reasoned that impaired membrane binding in Dyn1-R361S and Dyn1-R399A could be due to a perturbation in dynamin quaternary structure that significantly reduces its avidity for the target membrane.
To test whether Dyn1-R361S and Dyn1-R399A have disrupted quaternary structures in their unassembled state, we initially determined their sedimentation coefficients (S) by sedimentation velocity analytical ultracentrifugation. In contrast to Dyn1-WT, which sedimented at ∼10–12 S, both Dyn1-R361S and Dyn1-R399A sedimented predominantly at ∼6 S with only minor fractions at 10–12 S corresponding to that of the WT (Figure 2A). These data indicated that Dyn1-R361S and Dyn1-R399A are indeed disrupted in quaternary structure with the equilibrium shifted in favor of smaller species, presumably monomers or dimers.
Figure 2. Dyn1-R361S and Dyn1-R399A are assembly-defective dimers. (A) Sedimentation coefficients (S) of Dyn1-WT (solid line), Dyn1-R361S (short dashes) and Dyn1-R399A (long dashes) at 5 μM each were obtained as described under Materials and methods and are plotted here as a function of the relative concentration of each species. (B) SEC elution profiles for Dyn1-WT (solid line), Dyn1-R361S (short dashes) and Dyn1-R399A (long dashes) run through a Superose 6 10/30 HR (Amersham Biosciences) column were obtained as described under Materials and methods. Protein absorbance at 280 nm (A280) in milli-absorbance units (mAU) is plotted here as a function of elution volume (ml). Arrowhead points to the elution of dynamin microaggregates in the void volume.
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Accurate determination of the quaternary structure of Dyn1-R361S and Dyn1-R399A by sedimentation equilibrium analytical ultracentrifugation, as determined previously for Dyn1-WT (Muhlberg et al, 1997), was precluded owing to a slow rate of dynamin microaggregation in our samples (see void volume in Figure 2B). To overcome this limitation, both WT and mutant dynamin samples were resolved by size-exclusion chromatography (SEC; Figure 2B) and the major species in the included volume were analyzed by on-line multiangle laser light scattering (MALLS), a technique now widely used for unambiguous molecular mass determinations (Wen et al, 1996; Andersson et al, 2003; Ye, 2006). Unlike SEC, which relies on data based on relative standards and is influenced by molecular shape or sedimentation equilibrium analytical ultracentrifugation, which is time consuming resulting in protein aggregation during the course of the experiment, this combination of SEC and MALLS (SEC-MALLS) is relatively quick and determines the average molecular mass of each eluted fraction directly without calibration against standards or assumptions of molecular shape and size. SEC-MALLS data analyses unambiguously showed that Dyn1-R361S and Dyn1-R399A are predominantly dimers in contrast to Dyn1-WT tetramers (Table I). Thus, these data reveal that R361 and R399 in the middle domain are critical for establishing dynamin tetrameric structure in the unassembled state.
Dyn1-R361S and Dyn1-R399A exhibit impaired cooperativity in self-assembly
Previous studies have shown that dynamin's GTPase activity increases nonlinearly or sigmoidally with increasing protein concentration, reflecting positive cooperativity in higher-order dynamin–dynamin interactions that disproportionately enhances GTPase activity (Warnock et al, 1996; Song et al, 2004b). To determine whether the assembly defect in Dyn1-R361S and Dyn1-R399A is due to impaired positive cooperativity, we determined their GTP hydrolysis rates with increasing protein concentration and at low ionic strength (no salt) that favors higher-order self-assembly. Whereas the GTP hydrolysis rates of Dyn1-WT increased sigmoidally with increasing protein concentration, as expected, only a linear response was elicited from Dyn1-R361S and Dyn1-R399A, indicating impaired positive cooperativity in dynamin–dynamin interactions that promote higher-order self-assembly (Figure 3).
Figure 3. Dyn1-R361S and Dyn1-R399A dimers exhibit impaired cooperativity in self-assembly. GTP hydrolysis rates of Dyn1-WT (○), Dyn-R361S (▪) or Dyn1-R399A (⧫) at low ionic strength (no salt) were determined at 37°C as described under Materials and methods, and are plotted as a function of dynamin concentration. The GTPase activities of Dyn1-R361S and Dyn1-R399A are linearly proportional to their concentration in contrast to Dyn1-WT, which exhibits sigmoidal behavior. The average values of at least three independent experiments are shown along with standard deviations.
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Dynamin's basal GTPase activation is not rate-limited by nucleation
In recent studies using a coupled assay to measure the kinetics of GTP hydrolysis of either the yeast mitochondrial dynamin-related protein, Dnm1, or Dyn1-WT, a kinetic ‘lag’ was observed upon dilution of these proteins into low ionic strength buffer to trigger self-assembly before steady-state GTP hydrolysis rates were achieved (Ingerman et al, 2005; Sever et al, 2006). The duration of the lag decreased with increasing protein concentration, suggesting that it represented a rate-limiting nucleation event also cooperative with respect to GTP concentration. Consistent with this interpretation, no lag was detected when assaying middle domain mutants, Dnm1G385D or Dyn1-I690K, which coincidentally also exist as assembly-defective dimers in solution (Ingerman et al, 2005; Sever et al, 2006).
We have used this coupled assay to examine the kinetics of basal GTP hydrolysis by Dyn1-WT in comparison to Dyn1-R399A, which exhibited the greatest defect in self-assembly (Figures 1A, B and 3). Interestingly, we also detected a pronounced kinetic lag for Dyn1-WT even when assayed at physiological ionic strength (150 mM KCl), a condition that does not favor dynamin self-assembly in the absence of an appropriate lipid template (Figure 4A and Table II). In contrast to previous studies conducted at low ionic strength, we also detected a lag with the assembly-defective Dyn1-R399A mutant (Figure 4B and Table II). No lag was detected when the dynamin GTPase domain alone (residues 1–307) was subjected to this assay, indicating that it is not an artifact of assay conditions and also suggesting that the molecular events occurring during the lag phase involve domains other than or in addition to the GTPase (Supplementary Figure 3).
Figure 4. Activation of dynamin basal GTPase is not due to nucleation. (A, B) The kinetics of basal GTP hydrolysis in Dyn1-WT (○) or Dyn1-R399A (⧫) at 2, 4 and 6 μM protein concentration was measured at 37°C using the GTP regenerating system as described under Materials and methods. The concentration of GTP hydrolyzed and released is plotted as a function of time. The average values of at least three independent experiments are shown. (C, D) Time-dependent emission intensity profiles for 0.1 μM Dyn1-WTBODIPY (○) and Dyn1-R399ABODIPY (⧫) before and upon addition (arrowhead) of either GTP (1 mM final) or liposomes (30 μM final) were obtained as described under Materials and methods. BODIPY was excited at 490 nm and emission was monitored at 510 nm. F0 is the initial intensity of BODIPY at time ‘0’ and F is its intensity at time ‘t’.
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Table 2. Kinetic characterization of Dyn1-WT and Dyn1-R399A
|Kinetic lag (min)a|
| || || |
|Steady-state basal specific activity (min−1)b|
If this lag represents a rate-limiting nucleation event as suggested for dynamin at low ionic strength (Ingerman et al, 2005; Sever et al, 2006), and if the steady-state basal rate of GTP hydrolysis at physiological ionic strength is also limited by dynamin–dynamin higher-order interactions, then one would expect a substantial reduction in the lag period and an exponential increase in the rate of basal GTP hydrolysis with increasing protein concentration. Contrary to this scenario, there was no significant decrease in the lag period for either Dyn1-WT or Dyn1-R399A with increasing protein concentration (Table II). In addition, the specific activity of Dyn1-WT merely doubled with a three-fold increase in protein concentration (from 2 to 6 μM) showing a less than linear dependence on it (Table II). Similarly, Dyn1-R399A, which had slightly higher rates of basal GTP hydrolysis than Dyn1-WT, exhibited only a modest concentration-dependent increase in its specific activity (Table II). These data suggest that the basal GTPase activity of dynamin at physiological ionic strength is independent of higher-order protein–protein interactions.
To directly probe for evidence of higher-order dynamin–dynamin interactions under these conditions, we developed and employed a fluorescence-based assay that monitors dynamin self-assembly in real time. Here, Dyn1-WT and Dyn1-R399A were both labeled with the thiol-reactive BODIPY dye (BODIPY-FL C1-IA, hereafter shortened to BODIPY) that selectively and covalently modifies reactive Cys residues on the protein surface (out of a total of seven). As statistical proximity of two excited-state BODIPY fluorophores within a critical radius of ∼25 Å leads to self-quenching (Dahim et al, 2002; Fernandes et al, 2003), the potential decrease in BODIPY fluorescence intensity upon DynBODIPY–DynBODIPY interactions was used as an index of dynamin self-assembly. Control experiments established that BODIPY labeling did not alter the GTPase activity or self-assembly properties of the respective proteins (data not shown). As expected, self-assembly of Dyn1-WTBODIPY on PI4,5P2-containing membranes resulted in nearly 15% quenching of BODIPY emission intensity, whereas, on the other hand, incubation of Dyn1-R399ABODIPY with these membranes did not show any significant quenching, also consistent with the defect in its higher-order self-assembly (Figure 4C). The apparent lack of ordered Dyn1-R399A self-assembly upon incubation with PI4,5P2-containing membranes (Figures 1C and 4C) also correlated well with the nearly 90% reduction in its stimulated GTPase activity despite 50% binding to these membranes (Figure 1B).
Using this assay, we then determined whether dynamin self-assembly or nucleation ensued upon GTP addition. Upon GTP addition, neither Dyn1-WTBODIPY nor Dyn1-R399ABODIPY showed any significant quenching of BODIPY emission intensity, clearly indicating lack of GTP-dependent nucleation or higher-order self-assembly under these conditions (Figure 4D). From these data, we conclude that the activation of the dynamin GTPase observed before steady-state basal GTP hydrolysis is not due to dynamin–dynamin higher-order interactions or nucleation, but perhaps to a slow, allosteric conformational transition in unassembled dynamin relayed between subunits of a tetramer, or a dimer in the case of Dyn1-R399A, presumably by the middle domain and/or the GED that participate in subunit–subunit interactions.