(Received for publication, March 18, 1997, and in revised form, May 28, 1997)
From the Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, New York 12201-0509 and the Department of Biomedical Sciences, State University of New York, Albany, New York 12201-0509
As a molecular motor, dynein must coordinate ATP
hydrolysis with conformational changes that lead to processive
interactions with a microtubule and generate force. To understand how
these processes occur, we have begun to map functional domains of a dynein heavy chain from Dictyostelium. The
carboxyl-terminal 10-kilobase region of the heavy chain encodes a
380-kDa polypeptide that approximates the globular head domain.
Attempts to further truncate this region fail to produce polypeptides
that either bind microtubules or UV-vanadate cleave, indicating that
the entire 10-kilobase fragment is necessary to produce a properly
folded functional dynein head. We have further identified a region just
downstream from the fourth P-loop that appears to constitute at least
part of the microtubule-binding domain (amino acids 3182-3818). When
deleted, the resulting head domain polypeptide no longer binds
microtubules; when the excised region is expressed in
vitro, it cosediments with added tubulin polymer. This
microtubule-binding domain falls within an area of the molecule
predicted to form extended -helices. At least four discrete sites
appear to coordinate activities required to bind the tubulin polymer,
indicating that the interaction of dynein with microtubules is
complex.
In eukaryotic cells, dynein is a ubiquitous high molecular mass ATPase that moves organelles and other cellular cargo toward the minus ends of microtubules (1, 2). The globular head domain, largely encoded by the dynein heavy chain (DHC)1 gene, couples ATP hydrolysis and microtubule binding to generate conformational changes that provide force for this movement (3). Within the DHC, four P-loop motifs partially identify sites for nucleotide binding. The first of these motifs is highly conserved among cytoplasmic and axonemal dyneins and represents the major ATP catalytic site for force production (1, 4-7). The other three P-loops likely bind nucleotide (8), but their contribution to the mechanochemical cycle of dynein is so far unknown. The region of the heavy chain responsible for microtubule binding has not yet been identified. A conventional mapping strategy produces increasingly smaller fragments and tests these for relevant activity in vitro or in vivo. However, soluble expression of reasonably sized DHC fragments (i.e. >100 kDa) in prokaryotic systems is problematic. Even eukaryotic expression is not straightforward. Because the DHC provides an essential function in some organisms and activities (9-16), expression of constructs that retain partial activity could be toxic.
For both kinesin and myosin motors, the regions believed to interact with their respective filaments are located fairly close to the catalytic P-loop (17-20). It is possible that dynein follows a similar design with a single microtubule binding domain near the primary P-loop. We previously characterized the in situ expression of a 107-kDa fragment of the DHC that contained the first two P-loop motifs (21). Although a small amount of polypeptide appeared to cosediment with microtubules when analyzed by immunoblots, this was substantially less than the native heavy chain and clearly does not represent a native binding activity. This argues that the nucleotide-sensitive interaction of dynein with microtubules is not self-contained within a simple region flanking the first P-loop. Alternatively, there may be weak affinities associated with each of the four P-loop motifs, and microtubule binding is a cooperative effort involving multiple regions. Finally, the microtubule-binding domain may be located elsewhere in the globular head. An important prerequisite for its placement is that binding must be mechanically coupled to ATP catalysis to allow for cyclical on/off interactions.
We describe here efforts to express a series of DHC gene constructs in
Dictyostelium. This work roughly defines a minimal functional unit for the dynein motor domain. We further demonstrate that a region centered around a predicted two-part -helical domain of the heavy chain, just downstream of the fourth P-loop, is able to
bind microtubules in vitro. When this domain is deleted, a dynein head fragment expressed in vivo no longer interacts
with microtubules and loses its ability to undergo a UV-vanadate
cleavage reaction. The sequence encoding this motif is complex and may require substantial secondary structure for its activity. This work
identifies an important structural domain of dynein and links this
activity to ATP hydrolysis.
Most of the molecular manipulations and culture conditions have been previously described (21, 22). Briefly, fragments of the dynein heavy chain gene from Dictyostelium were ligated between the DHC promoter and an actin 8 transcriptional terminator on a plasmid containing a G418 resistance marker. Constructs were introduced into AX-2 Dictyostelium cells by CaPO4 precipitation of supercoiled plasmid DNA (23, 24). Colonies were selected for growth in 10 µg/ml G418 and cloned as described previously (21).
Biochemical MethodsGeneration of Dictyostelium high speed supernatants, microtubule affinity, UV cleavage, electrophoresis, and immunoblotting were performed as described in Ref. 25. Bovine tubulin was isolated and purified essentially as described in Refs. 26 and 27. Purified rabbit skeletal muscle actin was purchased from Cytoskeleton (Denver, CO).
In Vitro Transcription/TranslationFor in vitro expression, an initial EcoRI fragment of the DHC gene (encoding amino acids 3182-3679) and several subfragments were cloned into the pET 5 series of expression vectors (28), just downstream from the T7 RNA polymerase binding site. Products were expressed in vitro using a coupled transcription/translation reticulocyte lysate (Promega Corp., Madison, WI). 50-µl reaction mixtures were assembled following the manufacturer's instructions, using [35S]methionine to visualize newly synthesized polypeptides. After a 75-min synthesis at 30 °C, samples were diluted 5-10-fold with PMEG buffer (100 mM PIPES, pH 7.0, 5 mM EGTA, 0.1 mM EDTA, 4 mM MgCl2, 0.9 M glycerol) and clarified at 80,000 × g for 15 min at room temperature in a Beckman TLA 100.2 rotor. Supernatants were removed and either supplemented with 0.5 mg/ml taxol-stabilized tubulin or an equivalent volume of PMEG/taxol buffer for a precipitation control. In two sets of experiments, additional samples were incubated with approximately 0.4 mg/ml of purified actin. After a 15-min incubation at room temperature, the samples were underlaid with an equal amount of 20% sucrose in PMEG/taxol and spun again. Supernatant and pellet fractions were saved and electrophoresed on 12.5% acrylamide gels. After destaining, the gels were incubated in Amplify (Amersham Corp.), dried, and exposed to film.
The Dictyostelium DHC encodes an open reading frame of 4725 amino acids (6). Previous work showed that a 380-kDa carboxyl-terminal fragment corresponds to a single globular head (21). This fragment binds to microtubules in an ATP-sensitive fashion indistinguishable from native dynein and undergoes a UV-vanadate cleavage, a reaction diagnostic for dynein and for a structurally active ATP catalytic domain (29).
Despite using the same general construct design and following identical
transformation and cloning procedures, efforts to express smaller units
of the mechanochemical head in Dictyostelium had mixed
results. As shown in Fig. 1, some constructs express quite well, whereas others do not. Partial head domains that encode the
central region are problematic. In general, these correlate with poor
transformation efficiencies and fail to produce detectable product.
Only the 270-kDa plasmid expresses a polypeptide of predicted molecular
mass, albeit at a level significantly less than the native heavy chain.
In contrast, transformation efficiencies are consistently good for
plasmids that lack the central head region (107, 115, and 318 kDa), and
cells produce a robust amount of material. However, none of these
partial head domain fragments show good microtubule binding or
UV-vanadate cleavage (Fig. 2 and data not shown).
The inability to produce some DHC fragments suggests that these
polypeptides are either unstable or toxic. None of the head domain
fragments that do express well show substantial dynein-like activity.
Therefore it seems reasonable to predict that the difference between
these two sets of constructs (i.e. the central head region) contains an activity important for dynein's structure or function. An
SphI restriction digest of the DHC gene removes an in-frame 1.6-kilobase fragment of sequence (amino acids 3105-3643) within this
region. This excised segment encodes a structurally unique region of
the heavy chain, containing two predicted -helical motifs. Fig. 2
shows that AX-2 cells transformed with the 380K
Sph construct
abundantly produce the 318-kDa polypeptide. However, unlike either
native dynein or the 380-kDa head domain fragment, this 318-kDa
polypeptide does not cosediment with microtubules in binding assays and
fails to cleave in the presence of UV light and vanadate, indicating
that the ATP catalytic site is no longer active.
Deletion of this 1.6-kilobase fragment
from the DHC gene could sufficiently perturb secondary structure to
affect a microtubule-binding domain located elsewhere in the DHC
sequence. Although we cannot rigorously exclude this possibility, we
can demonstrate in vitro that the excised fragment encodes a
microtubule binding activity. Figs. 3 and
4 summarize this in vitro expression work. A
1.5-kilobase construct encoding 57 kDa was expressed in a reticulocyte
lysate and then incubated with purified bovine microtubules. The
resulting polypeptide pelleted through a sucrose cushion in the
presence of microtubules but not appreciably in their absence,
suggesting it binds to the taxol-stabilized polymer. Polypeptides
encoded by the helix-1 domain (20 kDa), the central region (30 kDa), or the helix-2 domain (24 kDa) did not appreciably pellet in this assay.
However, combining helix-1 + the central region (43 kDa), helix-2 + the
central region (44 kDa), or helix 1 + 2 (lacking the central region, 39 and 45 kDa) substantially increased this binding activity. The region
just downstream from helix 2 (16 kDa) also showed a weak affinity for
microtubules. Combining the 16-kDa fragment with helix-2 (40 kDa) and
with helix-2 + the central region (60 kDa) produced polypeptides with
progressively greater binding activity.
The buffer alone control presented for each construct indicates that
pelleting is not merely due to protein aggregation. However, because
tubulin is an acidic polymer (pI 5.5), it is possible that the in
vitro expressed polypeptides nonspecifically interact through
charged residues. Actin also forms long polymers and carries a net
overall charge similar to that of tubulin (pI. 5.4) (30). Fig.
5 compares the sedimentation of the 57- and the 30-kDa
polypeptides in the presence of molar excesses of microtubules and
actin filaments. Although actin sedimentation results in a slight
increase of pelletable 57-kDa product over buffer alone, there is
substantially more polypeptide in the tubulin pellet. The 30-kDa
polypeptide does not pellet in the presence of microtubules nor in the
actin polymer control. These results strengthen the argument that the
interactions described here are tubulin-specific.
The results presented in Figs. 3, 4, 5 indicate that this predicted helical region of the DHC contains an ability to cosediment with tubulin and thus may at least in part define the microtubule-binding domain of dynein. They further indicate that this region is complex and binding requires either substantial secondary structure or that multiple contact sites with the polymer are involved. In the in vitro pelleting assays, only a small percentage of the total expressed polypeptide appears to cosediment with microtubules. Although this is consistent with what we have found in Dictyostelium high speed supernatants, where only a fraction of the total native DHC cosediments with bovine microtubules,2 it may also indicate a partial microtubule binding ability. Native dynein contains both high and low affinity microtubule binding states, which are likely the product of geometrical changes within the microtubule-contact site. Without detailed information on the tertiary structure of this region in both the native molecule and the fragments expressed here, quantitative assessments on the binding efficiency are probably not meaningful.
The work presented here describes two related efforts to understand the domain structure of a dynein heavy chain gene. The first describes efforts to express functional units of the globular head domain, and the second identifies a region of the molecule that appears to contain a microtubule binding activity.
A Minimal Motor Domain for DyneinBiochemical and structural data indicate that a 380-kDa carboxyl-terminal fragment of the Dictyostelium DHC corresponds to a functionally active, single globular head of dynein (21). The data presented here describe efforts to produce substantially smaller head domain fragments that retain an ATP-sensitive microtubule binding activity. In our hands, several constructs truncated from the carboxyl-terminal end fail to produce detectable product, indicating that the specified polypeptide either is highly unstable or is toxic to the cell. Of the smaller constructs that are produced in vivo, none show a substantial ability to bind microtubules and, for the ones containing the first P-loop, fail to undergo the UV-vanadate cleavage normally associated with this motif (29). Truncations in from the amino terminus of the head domain would remove the P-loop associated with ATP hydrolysis and thus would fail to make complete motors.
A deletion from the middle of the head domain produces a polypeptide that appears stable but does not interact with microtubules or UV-vanadate cleaves. Moreover, the region excised contains an ability to bind microtubules in vitro. If an ATP-insensitive microtubule binding activity is retained in constructs expressed in vivo, then these polypeptides would likely coat the surface of the microtubules, inhibit dynamics and associated organelle motility, and likely lead to cell death. This may at least partially explain our difficulty in expressing some of the head domain constructs.
Although the first P-loop in the DHC sequence plays a prominent role in
binding the nucleoside triphosphate, additional residues are necessary
for ATP hydrolysis (31). Kinesin and myosin share at least three other
motifs in common with some GTPases that fold together forming the ATP
catalytic domain (reviewed in Ref. 32). Although identical sequences
are not obvious in the Dictyostelium DHC, it seems
reasonable to predict that similar structural motifs are required for
dynein; these could be distributed anywhere along the linear sequence.
Thus even though the Sph truncation occurs over 1100 amino acids
downstream of the first P-loop, the inability of the 318-kDa
polypeptide to UV-cleave indicates that the catalytic pocket is not
folded properly or is missing a functional element necessary for ATP
hydrolysis. Together with the data discussed in the previous
paragraphs, these results suggest that the entire 380-kDa
carboxyl-terminal fragment is necessary for ATP-sensitive microtubule
binding activity and thus roughly defines the minimal functional head
domain for dynein.
The in
vivo deletion and the in vitro expression work suggest
there are microtubule binding activities located within the predicted
-helical region just after the fourth P-loop in the DHC. A
preliminary report describing a similar finding for another cytoplasmic
dynein has also recently appeared (33). Comparative sequence analysis
shows this region to be well conserved among dynein heavy chains.
Within this region, the Dictyostelium sequence is
approximately 50% identical to most cytoplasmic dyneins and close to
30% identical to axonemal dyneins sequenced to date. Our work suggests
that at least four discrete units can act in different combinations to
mediate the binding of dynein to microtubules. Comparisons do not show
an obvious sequence conservation between these four domains, suggesting
that they do not contain simple repeated motifs similar to other
microtubule-binding proteins, e.g. MAP-2, MAP-2C, MAP-4, and
tau (34).
The predicted -helical regions just after the fourth P-loop
represent a unique structural domain in the dynein molecule. The
borders of these regions are arbitrarily defined here by proline residues. Although the true structure of this domain has yet to be
determined, the position and spacing of the proline residues are well
conserved among axonemal and cytoplasmic dyneins. Previous discussions
of functional properties for this domain include: 1) the helices could
form a projection off the head domain and provide the ATP-sensitive
B-link to the adjacent microtubule found in several axonemes (4, 35);
2) they may combine with another, shorter predicted helical domain
preceding the first P-loop and participate in forming the tertiary
structure of the head domain or the dynein molecule (1, 36); 3) they
may form coiled-coil interactions with other polypeptides (4, 36); and
4) may play a role in the conformational changes associated with force production (37). Our data strengthen the arguments presented for 1) and
4). If the region does form two or more helical domains, they could
serve to project a microtubule contact site or could serve as
mechanical levers to raise/lower a binding domain in conjunction with
structural changes produced by ATP catalysis.
A microtubule-binding sequence motif has been proposed in common with dyneins and kinesins (38, 39). P-X6E-X4-L represents the core consensus sequence of this motif and is surrounded by several conserved hydrophobic, polar, and charged regions (39). This sequence was described just after the first P-loop, about the same distance downstream in dynein, kinesin, and kinesin-like proteins. Although it falls within a region of kinesin shown experimentally to bind microtubules (17), a similar relationship to functional activity within dynein has not been reported. Interestingly, this core motif is also found within the domains we suggest have microtubule binding activity. Prolines 3198, 3366, 3648, and 3712 are followed by a glutamic acid 6 residues downstream and a leucine (or in one case, isoleucine) 3-5 residues further. The latter three of these "motifs" are well conserved among cytoplasmic dyneins.
Here we present data suggesting that the entire carboxyl-terminal
two-thirds of the dynein heavy chain from Dictyostelium is
essential to comprise a functional head domain. We also suggest that
there are at least four domains, each containing approximately 125 amino acids, clustered around a predicted -helical region downstream
from the fourth P-loop that collectively participate in binding the
motor to the microtubule polymer. Data also exist to suggest that
dynein binds to both
- and
-tubulin (40), and structure work
indicates that the dynein head is large enough to interact with
multiple dimers. If a single dynein can bind to both
- and
-tubulin, the contact regions would not necessarily be identical.
However, they might contain a similar core structure that coordinates
affinity with ATP hydrolysis. Taking this together, we propose that
cytoplasmic dynein makes more than one functional contact with a
microtubule. This could be important in binding (does it dock to or
does it grip the tubulin surface) or during its mechanochemical stroke
(instead of rocking a lever arm, does the head roll?). At a minimum,
this work associates a relatively defined region of the DHC with a
functional activity and will permit a more focused analysis of how
dynein binds microtubules.
We thank Irina Tikhonenko, Theresa Church, and Tina Fortier for technical assistance and cell culture and Drs. Alexey Khodjakov, Dan Rosen, and Conly Rieder for comments on the manuscript. Dr Stephen King kindly suggested the actin filament control. A special thanks and acknowledgment goes to Dr. Khodjakov and the Wadsworth Center Video Light Microscopy Core for assistance in preparing some of the figures.