A structural model reveals energy transduction in dynein.
ABSTRACT Intracellular active transport is driven by ATP-hydrolyzing motor proteins that move along cytoskeletal filaments. In particular, the microtubule-associated dynein motor is involved in the transport of organelles and vesicles, the maintenance of the Golgi, and mitosis. However, unlike kinesin and myosin, the mechanism by which dynein converts chemical energy into mechanical force remains largely a mystery, due primarily to the lack of a high-resolution molecular structure. Using homology modeling and normal mode analysis, we propose a complete atomic structure and a mechanism for force generation by the motor protein dynein. In agreement with very recent electron microscopy (EM) reconstructions showing dynein as a ring-shaped heptamer, our model consists of six ATPases of the AAA (ATPases associated with various cellular activities) superfamily and a C-terminal domain, which is experimentally known to control motor function. Our model shows a coiled coil spanning the diameter of the motor that accounts for previously unidentified structures in EM studies and provides a potential mechanism for long-range communication between the AAA domains. Furthermore, normal mode analysis reveals that the subunits of the motor that contain the nucleotide binding sites exhibit minimal movement, whereas the rest of the motor is very mobile. Our analysis suggests the likely domain rearrangements of the motor unit that generate its power stroke. This study provides insights into the structure and function of dynein that can guide further experimental investigations into energy transduction in dynein.
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ABSTRACT: Ferritin-like molecules show a remarkable combination of the evolutionary conserved activity of iron uptake and release that engage different pores in the conserved ferritin shell. It was hypothesized that pore selection and iron traffic depend on dynamic allostery with no conformational changes in the backbone. In this study, we detect the allosteric networks in Pseudomonas aeruginosa bacterioferritin (BfrB), bacterial ferritin (FtnA), and bullfrog M and L ferritins (Ftns) by a network-weaving algorithm (NWA) that passes threads of an allosteric network through highly correlated residues using hierarchical clustering. The residue-residue correlations are calculated in the packing-on elastic network model that introduces atom packing into the common packing-off model. Applying NWA revealed that each of the molecules has an extended allosteric network mostly buried inside the ferritin shell. The structure of the networks is consistent with experimental observations of iron transport: The allosteric networks in BfrB and FtnA connect the ferroxidase center with the 4-fold pores and B-pores, leaving the 3-fold pores unengaged. In contrast, the allosteric network directly links the 3-fold pores with the 4-fold pores in M and L Ftns. The majority of the network residues are either on the inner surface or buried inside the subunit fold or at the subunit interfaces. We hypothesize that the ferritin structures evolved in a way to limit the influence of functionally unrelated events in the cytoplasm on the allosteric network to maintain stability of the translocation mechanisms. We showed that the residue-residue correlations and the resultant long-range cooperativity depend on the ferritin shell packing, which, in turn, depends on protein sequence composition. Switching from the packing-on to the packing-off model reduces correlations by 35%-38% so that no allosteric network can be found. The influence of the side-chain packing on the allosteric networks explains the diversity in mechanisms of iron traffic suggested by experimental approaches.The Journal of Chemical Physics 03/2014; 140(11):115104. · 3.12 Impact Factor
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ABSTRACT: Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.Biophysical Journal 08/2014; 107(3):662–671. · 3.83 Impact Factor
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ABSTRACT: Spermatozoa are highly specialized cells. Adenosine triphosphate (ATP), which provides the energy for supporting the key functions of the spermatozoa, is formed by 2 metabolic pathways, namely glycolysis and oxidative phosphorylation (OXPHOS). It is produced in the mitochondria through OXPHOS as well as in the head and principal piece of the flagellum through glycolysis. However, there is a great discrepancy as to which method of ATP production is primarily utilized by the spermatozoa for successful fertilization. Mitochondrial respiration is considered to be a more efficient metabolic process for ATP synthesis in comparison to glycolysis. However, studies have shown that the diffusion potential of ATP from the mitochondria to the distal end of the flagellum is not sufficient to support sperm motility, suggesting that glycolysis in the tail region is the preferred pathway for energy production. It is suggested by many investigators that although glycolysis forms the major source of ATP along the flagellum, energy required for sperm motility is mainly produced during mitochondrial respiration. Nevertheless, some studies have shown that when glycolysis is inhibited, proper functioning and motility of spermatozoa remains intact although it is unclear whether such motility can be sustained for prolonged periods of time, or is sufficiently vigorous to achieve optimal fertilization. The purpose of this article is to provide an overview of mammalian sperm energy metabolism and identify the preferred metabolic pathway for ATP generation which forms the basis of energy production in human spermatozoa during fertilization.Asian Journal of Andrology 11/2014; · 2.14 Impact Factor
A structural model reveals energy transduction
Adrian W. R. Serohijos*, Yiwen Chen*, Feng Ding†, Timothy C. Elston‡§, and Nikolay V. Dokholyan†§
Departments of *Physics and Astronomy,†Biochemistry and Biophysics, and‡Pharmacology, University of North Carolina, Chapel Hill, NC 27599
Edited by Charles S. Peskin, New York University, New York, NY, and approved October 12, 2006 (received for review April 7, 2006)
Intracellular active transport is driven by ATP-hydrolyzing motor
proteins that move along cytoskeletal filaments. In particular, the
microtubule-associated dynein motor is involved in the transport
of organelles and vesicles, the maintenance of the Golgi, and
mitosis. However, unlike kinesin and myosin, the mechanism by
which dynein converts chemical energy into mechanical force
remains largely a mystery, due primarily to the lack of a high-
resolution molecular structure. Using homology modeling and
normal mode analysis, we propose a complete atomic structure
In agreement with very recent electron microscopy (EM) recon-
structions showing dynein as a ring-shaped heptamer, our model
consists of six ATPases of the AAA (ATPases associated with
various cellular activities) superfamily and a C-terminal domain,
which is experimentally known to control motor function. Our
model shows a coiled coil spanning the diameter of the motor that
accounts for previously unidentified structures in EM studies and
provides a potential mechanism for long-range communication
between the AAA domains. Furthermore, normal mode analysis
reveals that the subunits of the motor that contain the nucleotide
binding sites exhibit minimal movement, whereas the rest of the
motor is very mobile. Our analysis suggests the likely domain
rearrangements of the motor unit that generate its power stroke.
This study provides insights into the structure and function of
dynein that can guide further experimental investigations into
energy transduction in dynein.
AAA ? homology modeling ? molecular motors ? electron microscopy ?
microtubule-associated molecular motor dynein is involved in the
transport of organelles and vesicles, the maintenance of the Golgi,
and mitosis (2, 3). Mutations in this protein have been implicated
Unlike the motor proteins kinesin and myosin, the mechanism by
which dynein converts chemical energy into mechanical force
remains largely a mystery. The primary reason for this is the lack
of a high-resolution molecular structure. Cytoplasmic dynein is a
large multisubunit complex (1.2 MDa) composed of two heavy
chains (?0.5 MDa each) (6), making its structural characterization
extremely challenging. In this study, we use homology modeling to
propose a structure for the motor unit of the dynein heavy chain,
which is the site for energy transduction and force generation (see
Fig. 1a). Next, we perform normal mode analysis to determine the
large-scale motions of the protein. Together, these results suggest
a mechanism for both force generation and regulatory control in
Sequence analysis of dynein’s motor unit indicates that it
consists of six concatenated AAA (ATPases associated with
diverse cellular activities) subunits, an extended stalk that con-
tains a microtubule binding domain, and a C-terminal domain
that is twice the size of an AAA subunit (Fig. 1) (7, 8). Mocz and
Gibbons (8) used homology modeling to construct the first
structural model of dynein. Their model consisted of the six
AAA modules (AAA1–AAA6) arranged as a symmetric hex-
otor proteins use energy derived from ATP hydrolysis to
move along cytoskeletal filaments (1). In particular, the
amer (8). However, recent 3D EM reconstructions of negatively
stained cytoplasmic dynein from Dictyostelium discoideum show
the motor as a seven-lobe asymmetric ring (Fig. 1b) (9), with the
additional subunit presumably formed by the C domain. This
observation led Samso and Koonce (9) to propose a revised
structure in which the C domain was modeled as an extra AAA
subunit. Although the detailed structure of the C domain
remains unknown, there is increasing evidence that it plays a key
(10). For example, recent enzymatic analysis using recombinant
and proteolytic rat motor domain fragments suggests that the C
domain regulates ATPase activity (10). Interestingly, 3D EM
reconstructions of dynein also revealed additional structures
above and below the planar ring that are unaccounted for in
either previous model (Fig. 1b) (9). Corroborating these obser-
vations are 2D EM images of axonemal dynein showing a
stain-filled central cavity that results from a structure that is
neither an AAA module nor the C domain (11, 12). We show
below that this previously unidentified region is likely a coiled-
coil structure that spans the diameter of the motor ring and is
formed by the long interdomain region found between subunits
AAA5 and AAA6. We postulate that this extended chain
mediates long-range communication across the face of the
motor, in particular between domains AAA1–AAA4 and do-
Currently, several models are proposed for dynein’s power
stroke. In these models, ATP hydrolysis induces movement of
either the stalk, the tail or an flexible linker between the motor
unit and the tail (8, 9, 11, 13, 14). However, none of these models
directly predict how forces are transduced from the primary
hydrolytic site to the rest of the rest of the domains. From our
structural model and normal mode analysis, we propose a
detailed model of the conformational rearrangements in the
motor unit that lead to the dynein power stroke and a possible
mechanism for interdomain communication.
Modeling the Dynein Motor Unit. Using homology modeling, we
systematically constructed a complete structural model of the
includes the six AAA subunits, the linker regions that connect
structurally conserved units: an ??? Rossman fold subdomain
Author contributions: A.W.R.S., Y.C., F.D., T.C.E., and N.V.D. designed research; A.W.R.S.,
Y.C., F.D., T.C.E., and N.V.D. performed research; A.W.R.S., Y.C., F.D., T.C.E., and N.V.D.
analyzed data; and A.W.R.S., T.C.E., and N.V.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: PDB, Protein Data Bank.
Bank, www.pdb.org (PDB ID code 2GF8).
§To whom correspondence may be addressed. E-mail: email@example.com or telston@
This article contains supporting information online at www.pnas.org?cgi?content?full?
© 2006 by The National Academy of Sciences of the USA
December 5, 2006 ?
vol. 103 ?
no. 49 www.pnas.org?cgi?doi?10.1073?pnas.0602867103
and an ?-helical globular subdomain (Fig. 2a). Despite a ?20%
sequence identity between proteins in the AAA superfamily, up
to 50% of equivalent C?positions are within 2 Å rmsd (15). To
produce improved folds for the six AAA subunits of the dynein
motor unit, we used 3DJury (Methods) to search for candidate
homologs of these subunits and the linker regions that connect
them. 3DJury produces a consensus structure template based on
the results of multiple independent structure prediction algo-
rithms. The structural templates obtained in this way have
consensus scores well above the confidence threshold of 50,
which offers a prediction accuracy of 90% [see supporting
information (SI) Table 1, for complete bioinformatics results].
AAA domains and their adjacent linkers using the homology
modeling suite in Insight II (Methods). To evaluate the accuracy
of the models for each subunit, we compared the local environ-
ments of the residues in the predicted structures to the popu-
lation averaged residue environments determined from known
structures (SI Table 2). The profiles score show the current
model has better fold than earlier proposed models (8).
and a tail which oligomerizes with other dyneins. The atomic model of the motor unit contains six domains of the AAA enzyme family and a C domain. Domains
are colored and identified according to schema. Active ATP-binding sites are indicated by stars. (b) Model structure from homology fitted to a 25-Å EM map of
negatively stained cytoplasmic dynein (8) (EM map courtesy of M. Koonce). Motor unit consists of ‘‘smooth’’ (AAA1-AAA4) and ‘‘rough’’ (AAA5-C domain) sides.
Also shown are the linkers between AAA1 and AAA2 (light gray), between AAA2 and AAA3 (black), between AAA3 and AAA4 (light gray), and between AAA5
AAA5 and AAA6, whereas the smaller density may be the remnant of the tail fragment. (c) Model of the C domain consists of an ?–? toroid modeled from
complement component C3d (PDB ID code 1GHQ) and a ?-barrel modeled from leukemia-associated rhogef domain (PDB ID code 1TXD).
Model of the cytoplasmic dynein heavy chain. (a) Dynein consists of a motor unit (heavy chain) which generates force, a stalk that binds to microtubule,
(Inset) Putative site of primary hydrolysis with ATP docked. Walker A (i.e., P-loop) is red, Walker B is blue, Sensor 1 is yellow, and Sensor 2 is orange. (b) Sequence
alignment between the Dictyostelium discoideum AAA1 and template structure RuvB domain (PDB ID code 1HQC).
Model of the first AAA domain. (a) Canonical AAA fold (???-subdomain and all-? subdomain) with a synapomorphic pre-Sensor-1 ?-hairpin insertion.
Serohijos et al.
December 5, 2006 ?
vol. 103 ?
no. 49 ?
core, which is located between the first and second AAA
subunits, we docked an ATP molecule to the glycine-rich P-loop
(1969-GPAGTGKT-1976), which is the putative binding site for
the nucleotide phosphate tail (Fig. 2a). Within 5 Å of the docked
nucleotide, we found conserved residues in the Walker A and
Walker B motifs that bind the ? and ? NTP phosphates in all
P-loop NTPases. These conserved residues found in dynein
include K1975 in Walker A; D2021, E2022, and R2025 in Walker
B; and R2145 in Sensor 2 (Fig. 2a). These results are consistent
with recent biochemical studies showing the dynein mutant
K1975T trapped in a strong-binding state and devoid of motile
A notable structural feature found in some AAA enzymes
including dynein is a ?-hairpin insertion (denoted pre-Sensor-1
?-hairpin) in the ???-subdomain (Fig. 2a and SI Fig. 5). The
pre-Sensor-1 ?-hairpin insertion defines a phylogenetic superclade
that includes the Holliday junction migration motor protein RuvB
that was found to be a consensus template for subunits AAA1,
AAA3, and AAA5. In the current model, domains AAA1, AAA3,
and AAA5 are predicted to have ?-hairpin insertions in positions
2776, and 3743-RLGDQDVDF-3751, respectively. In RuvB, the
particular, mutations in the ?-hairpin of RuvB diminished its
functional interaction with RuvA, preventing branch migration of
the Holliday junctions (17, 18) . In dynein, the ?-hairpin insertions
may perform similar functions. For example, the flexibility of the
its proximity to the N-terminal tail suggest that the hairpin may be
crucial for coordinating AAA1 and tail movement.
To construct a structural model for the C domain, we followed
a protocol similar to the one used to construct models of the
AAA subunits. We found that the C domain’s first 290 residues
consist entirely of ?-helices, whereas the remaining 128-residue
stretch includes five ?-strands and terminates with a helix (Fig.
1c). We then determined a family of candidate proteins that
represent good structural templates for the two stretches of the
C domain. Interestingly, the candidate templates for the first 290
residues were structures of the complement component C3d
[Protein Data Bank (PDB) ID code 1GHQ], which attaches to
foreign antigens during immune response (19). Using the C3d
fragment as template, the first 290 residues acquired a dome-
shaped ?–? toroidal fold (19). The remaining five ?-strands and
last helix were built from the plecktrin homology (PH) domain
of the Leukemia-associated RhoGEF (PDB ID code 1TXD)
(20), which folds into a flattened seven-stranded ?-barrel capped
with a C-terminal helix. To obtain the complete structure of the
C domain, the two subdomains were docked together using rigid
body docking (see Materials and Methods). The ?-helical stretch
shows higher homology with its template structure than the
is performed by the more conserved ?-helical stretch.
Interestingly, the interdomain region between subunits AAA5
and AAA6 (denoted as IDR4) is 231 residues long, comparable
with the size of an AAA unit (whereas the length of the other
interdomain regions IDR1, IDR2, and IDR3 are 79, 103, and 92,
respectively) (Fig. 1). If IDR4 possesses a globular fold, then it
would manifest as an additional lobe in the reconstructed EM
densities (Fig. 1b) (9), and the motor would appear as an
octamer. On the other hand, one of the densities on the face
of the motor unit forms a long arch that spans the ring formed
by the AAA subunits (Fig. 1b), and is suggestive of a coiled coil.
The IDR4 is sufficiently long to span the ?8-nm facial density
of the motor unit. Moreover, coil prediction algorithms assign a
coiled-coil structure in the AAA5-IDR4 sequence, although the
length of the predicted coil varies for dyneins from different
species (SI Fig. 6). The search for structural homologs also
resulted in several coiled-coil structures. On the basis of these
results, we built IDR4 as a coiled coil using the cytoplasmic
domain of serine chemotaxis receptor (PDB ID code 1QU7)
(21) as a template.
The smaller lump on the face opposite the arching density (see
Fig. 1b and SI Fig. 7) could be the remnant of the dimerizing tail
tail has been labeled with antibody-Fab tag showed that the tag
is not rigid and can be found at various position around the
planar ring. The study suggests that the tail domain docks into
the center of the ring and that the tail sequence immediately
adjacent to the docking point is flexible. Thus, only the point of
attachment near of the tail will exhibit a density because the
flexible part will be averaged out, making the smaller facial
density (Fig. 1b) the more viable candidate for docking of the
Motor Unit Organization. EM reconstruction of the dynein motor
at 25 Å resolution from negatively stained D. discoideum dynein
shows a complex of seven densities arranged in an asymmetric
ring with ‘‘rough’’ and ‘‘smooth’’ edges (Fig. 1b) (9). The smooth
side consists of the more conserved AAA1–AAA4 domains,
resembling the symmetric oligomers of other large AAA com-
plexes with known crystal structures. The rough side of the
motor consists of AAA5, AAA6, and the C domain. Because the
sequences of these domains are less conserved, they are not
expected to follow the configuration of homomeric AAA com-
plexes, and, therefore, break the symmetry of the dynein motor
domain. This broken symmetry might play an important role in
effectively propagating force during the power stroke phase of
the hydrolysis cycle (see below).
To preserve functionally relevant interactions between domains
AAA1–AAA4 and to construct a regular tetramer for this portion
of the motor, we superimposed the models of these subunits onto
the ?54RNA polymerase activator NtrC1 (PDB ID code 1NY6)
(23). This protein has a known homogenous heptamer structure
the vector-quantization method implemented in SITUS (24) to fit
the AAA1–AAA4 tetramer to the EM density (Fig. 1b). To obtain
a preliminary orientation of the remaining domains, the atomistic
models of subunits AAA5, AAA6, and the C domain were fit
separately to their corresponding electron density lobes. We also
imposed the constraint that AAA5, IDR4, and AAA6 form a
continuous peptide. Thus, AAA5 was oriented such that its C
terminus faced the coiled coil. Similarly, AAA6 was oriented such
that its N terminus faced IDR4 (SI Text).
Finally, to arrive at the complete model, we docked IDR4 and
the rest of the interdomain regions to the seven domains using
a rigid-body docking protocol and shape complimentarity as
criteria. When the complete atomic model was refit to the EM
density of the entire motor unit, SITUS (25) ab initio identified
the correct orientation of the domains with a correlation of 0.74
(P ? 10?316; Fig. 1b; see SI Text for details).
Normal Mode Analysis of Global Motions. In the final model of the
motor unit, the interdomain regions and AAA units form a
compact backbone. The most closely packed part of the motor
found. We hypothesized that this compact structure is essential for
efficiently transducing forces generated at the ATP hydrolysis site
and is located between subunits AAA4 and AAA5. To investigate
motor unit’s dominant modes of motion (Fig. 3 and SI Fig. 8).
Normal mode analysis has been shown to accurately identify
structural sites that function as pivots and, therefore, can be used
to infer global motions of large molecular complexes (26). Normal
mode analysis also can be used to explore the intrinsic flexibility of
www.pnas.org?cgi?doi?10.1073?pnas.0602867103Serohijos et al.
molecular structures. Fig. 3a illustrates atomic displacements asso-
ciated with the three lowest frequency vibrational modes. The
of mode 1. From Fig. 3 (and the animations provided in SI Movies
and the C domain, whereas AAA1–AAA4 form a more compact
structure. These observations are made quantitative in Fig. 3b,
which lists the rmsd of the C? atoms of each subunit for the first
three normal modes.
In mode 1, the AAA5 subdomain exhibits an upward motion,
whereas AAA6 partially rotates about the IDR4 linker (Fig. 3a).
On the other hand, AAA1 to AAA4 and their linkers exhibit
minimal displacement. Interestingly, AAA5 is positioned at the
the dominant motion of the lowest frequency normal mode
occurs at the base of the stalk suggests that the stalk tilts during
the motor’s power stroke. Mode 2 is characterized by a
‘‘squeeze’’ applied to subunit AAA5 and the C domain coupled
with an outward motion by AAA6 (Fig. 3a). Similar to mode 1,
in mode 2, AAA1–AAA4 and their linkers exhibit minimal
movement. EM 3D reconstructions of the motor unit with stalks
positioned at 0°, 25°, and 45° relative to vertical show greatest
variation in electron densities corresponding to subunits AAA5
and AAA6 (9) (Fig. 3c). The direction and magnitude of the
domain displacements determined for modes 1 and 2 are con-
sistent with these observations (Fig. 3c). For example, the
motion predicted to occur in modes 1 and 2 is consistent with the
reorientation of subunit AAA6’s density observed for different
stalk positions (Fig. 3c).
The side views shown in Fig. 3 illustrate that the dominant
global motions of the molecule are not coplanar with the motor
ring. These results predict that any movement of the stalk or tail
will also contain a nonplanar component. A nonplanar bending
of the stalk is in agreement with static in situ tomography studies,
where stalks are shown to lie obliquely with respect to the dynein
ring (27). Moreover, this observation suggests that previous 2D
electron micrograph images in which the molecule is forced to lie
flat on the substrate might not accurately capture the confor-
mational changes that occur during force generation (27).
In mode 3, the C domain pushes on AAA1. It is not until mode
3 that the linker IDR4 intermediate to AAA5 and AAA6 moves
significantly, suggesting that IDR4 is a rigid structure. Similar to
modes 1 and 2, mode 3 produces an open-close movement at the
main catalytic site, which is found at the interface between
AAA1 and AAA2 (Fig. 3 and the animations in SI Movies 1–6).
In all three modes, the motion in the catalytic site is smaller
compared with the dominant motion in that mode. This obser-
vation suggests that, during the mechano-chemical events lead-
ing to force production, only minor conformational changes
occur in the catalytic site upon binding or release of ATP or
ADP. However, this small conformational change is amplified by
the rest of the motor domains (as shown below).
The first three normal modes show large movements of the
AAA1 pre-Sensor-1 ?-hairpin (Fig. 3a) despite minimal dis-
placements of the canonical AAA1 subdomains. Because the
?-hairpin projects from the rear of the AAA1 ?-? subunit (Figs.
1 and 3), small fluctuations in AAA1 are amplified by the
hairpin. This motion makes the hairpin a viable candidate for
coupling events in the ATP pocket of subunit AAA1 (the
shown to regulate dynein ATPase activity (10). Pre-Sensor-1
?-hairpins in other AAA modules of the motor unit may also
mediate interdomain interactions. In other AAA complexes that
contain pre-Sensor-1 ?-hairpins, this structure plays a significant
functional role. For example, in NtrC1, an AAA hexamer that
activates bacterial ?54-RNA polymerase holoenzyme, the ?-hair-
pin is used to bind ?54(28).
Discussion and Conclusion
The predicted structural model of the cytoplasmic dynein motor
unit consists of six AAA domains and a C-terminal domain
arranged in an asymmetric heptameric ring. The conserved
AAA1-AAA4 domains form a tetramer that is organized sim-
ilarly to other AAA homomer complexes. The less well con-
served AAA5, AAA6, and C-terminal domains constitute the
rough side of the motor. This asymmetric organization of the
motor complex is consistent with the postulated evolutionary
origin of the molecule in which the primordial homodimer pairs
AAA1–AAA2 and AAA3–AAA4 combined to form a tet-
of the backbone is rendered proportional to fluctuations of C?atoms. Arrows indicate the directions of dominant vibrations. AAA5, AAA6, and C domain exhibit the
most prominent variation in domain architecture in the three normal modes. (See SI Movies 1–6.) (b) Mean rmsd of C?in a domain, normalized by the largest
displacement and weighted by inverse frequency. (c) Superposition of reconstructed 3D structures of the motor unit in three distinct stalk conformations from EM
studies by Samso and Koonce (9). In the three stalk positions, the side formed by AAA5, AAA6, and C domain exhibit the largest variation (9).
Lowest frequency normal modes of dynein motor unit. (a) Superposition of two structures displaced in opposite directions along the normal mode. The size
Serohijos et al.
December 5, 2006 ?
vol. 103 ?
no. 49 ?
ramer, with the subunits AAA5 and AAA6 representing later
additions to the motor (14).
The ?-hairpin insertions inherited by AAA1, AAA3, and
AAA5 are unique and functionally important features of AAA
enzyme complexes in the PS1BH clade (21). In particular,
mutations in the ?-hairpin of RuvB abolish its physical and
functional interactions with the RuvA DNA recognition protein
(18). Because of its close proximity to the primary hydrolytic site
as well as the C-terminal and tail domains, the ?-hairpin likely
plays a role in coordinating the conformational changes involved
in the force generating mechano-chemical cycle. This hypothesis
can be tested by using site-directed mutations in the sequences
corresponding to the ?-hairpins.
linker that connects subunits AAA5 and AAA6. The model
spans the motor ring. In addition to contributing to the overall
rigidity of the motor, IDR4 provides a route for force propaga-
tion from the rigid smooth edge of the motor where the
nucleotide-binding sites are located to the flexible rough edge.
Specifically, IDR4 extends from AAA5, which is at the base of
the microtubule-binding stalk, to AAA3, whose nucleotide
binding pocket regulates the motor’s processivity (14, 16). The
IDR4 structure provides a clue to the important question of how
distant functional sites communicate with each other to generate
a coordinated mechano-chemical cycle.
Studies show that the ATP binding pocket in AAA3 is catalyt-
ically active (16, 29). How might this hydrolytic activity in AAA3
affect the microtubule-binding affinity of the stalk? AAA3 domain
has two subdomains (Fig. 5), an ??? subdomain and an ? subdo-
main, and the nucleotide binding P-loop is sandwiched between
induce the open and close movement between these subdomains.
Current models assume that perturbations in the AAA3 P loop are
propagated to AAA4, which is at the base of the microtubule-
binding stalk. Alternatively from our model, the open and close
movement in the AAA3 subdomains may be propagated through
the coiled coil to the AAA5, which is at the base of the stalk. The
orientation of the stalk’s globular tip likely dictates the stalks
Using 2D images of axonemal dynein, Burgess et al. suggest
the face of the ring (12). In another recent EM study, Meng et
al. (22) labeled the cytoplasmic dynein tail with an antibody-Fab
tag and used EM and single-particle image analysis. They found
the tag at various locations around the planar ring. Collectively,
the studies suggest that the tail could dock into the smaller facial
lump (Fig. 1b and SI Fig. 7) because the tail’s density will be
averaged out in the 3D EM reconstruction. In this model, both
the tail and IDR4 may interact with the AAA domains, and the
less flexible IC4 adds structural stability to the motor unit.
Our analysis of the three lowest frequency normal modes
indicates that large scale motions of the motor primarily involve
movements in subunits AAA5, AAA6, and the C domain,
whereas subunits AAA1–AAA4 function as a rigid structure.
This finding is consistent with recent observations from EM
reconstructed structures (Fig. 3c) (9, 30). We speculate that the
subunits AAA1–AAA4 provide the motor with a stationary
backbone against which forces generated in the primary catalytic
site can act. This generates conformational changes that prop-
agate sequentially through the C domain, AAA6 and AAA5 and
terminate with a movement of the microtubule-binding stalk
There are three current models for dynein’s power stroke. In
the first model, ATP causes a rotation of both the stalk and the
tail about the junctions that connect them (8, 9, 13). The second
model assumes that a conformational change of the tail swings
the motor unit and the stalk together (14). Lastly, the third
model assumes that a flexible structural linker between the
motor unit and tail bends upon coordinated conformational
rearrangements of the AAA domains (10). From our structural
model and normal mode analysis, model 2 is unlikely because of
the large motions in AAA5 to which the stalk is docked. We
propose the possible conformational rearrangements of the
domains movements within the motor unit that is the basis of
either model 1 or model 2 (Fig. 4). Binding or release of ATP
or ADP induces conformational change in the catalytic domain
between AAA1 and AAA2. Because of the rigid structure
formed by subunits AAA1-AAA4, the disturbance is propa-
gated in a clockwise direction through the C domain, AAA6, and
AAA5, causing the microtubule-binding stalk to flex. The
change in the angular position of the stalk possibly alters the
microtubule binding affinity of the stalk’s globular tip. These
with the findings in enzymatic studies of dynein domain frag-
ments suggesting that the stalk autoinhibits ATP?ADP release
in AAA1 and AAA3, and that the C domain also affects the
ATPase activity (10).
Using the same 380-kDa dynein fragment from which the EM
density is derived, Kon et al. labeled AAA2 and C domain with
BFP and the tail with GFP. They found that the dynein adopts
at least two conformational states, and the tail undergoes
ATP-induced motions relative to the motor domain during
transitions between the states (31). On the other hand, an EM
study where the tail fragment has been labeled with a FAB-
antibody tag, the tail did not have a preferred orientation (22).
This discrepancy in results may be due to a difference in
the nucleotide states. The resolution of this discrepancy warrants
further studies. In particular, we propose FRET studies using
constructs in which the tail, the motor domain, and the putative
coiled coil are labeled with fluorescent proteins.
Materials and Methods
Model Building. The sequence of the motor domain of cytosolic
dynein heavy chain of slime mold D. discodeum (GenBank
accession no. P34036) was submitted for threading to 3DJury
the AAA1, AAA2, and AAA4 were the Holliday junction
migration motor protein RuvB from Thermus thermophilus HB8
(PDB ID code 1HQC; ref. 18), clamp loader gamma complex of
Escherichia coli DNA polymerase III (PDB ID code 1JR3; ref.
34), and eukaryotic clamp loader (PDB ID code 1SXJ; ref. 35),
respectively. AAA3 and AAA5 were modeled from the same
hydrolytic sites (indicated by stars) induces conformational change that is
domain labels). These domain reorientations cause the stalk or tail to flex
about the junction that connects them to the motor unit, thus generating the
Model of power stroke. Binding ATP or release of ADP?Pi in the
www.pnas.org?cgi?doi?10.1073?pnas.0602867103Serohijos et al.
model used by AAA1. Similar to AAA2, AAA6 was built from
the clamp loader. The C domain is modeled from complement
C3d (PDB ID code 1GHQ; ref. 19) and pH domain of leukemia-
associated rhogef (PDB ID code 1TXD; ref. 20). We constructed
the atomic models using the Homology suite of INSIGHTII
(Accelrys, San Diego, CA). To arrange the domains in a
heptamer, we used QRANGE (24) and COLORES (25) (both
in SITUS; ref. 24) to fit the individual domain models to a EM
map of the dynein motor domain of D. discoideum (9). Finally,
we eliminated the clashes in the model structure by running
equilibrium simulations using a simplified protein model fol-
lowed by all-atom reconstruction (36, 37). The rmsd between the
models before and after reconstruction is 2.5 Å.
Normal Mode Analysis. Coordinates of the constructed model
were submitted to ELNemo server (http:??igs-server.
cnrs-mrs.fr?elnemo?index.html) (38) to compute the first 10
A detailed description of the model-building and refinement
and normal mode analysis can be found in SI Text.
We thank M. Koonce for generously providing the EM maps, B.
Temple for help and discussions on molecular modeling, and K. Wilcox
and S. Barton for a careful reading of the manuscript. This work was
supported in part by the Muscular Dystrophy Association Grant
MDA3720 (to N.V.D.), American Heart Association Grant 0665361U
(to N.V.D.), and National Institutes of Health Grant R01
GM078994-01 (to T.C.E.).
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