Structure of the complex of a mitotic kinesin with its
calcium binding regulator
Maia V. Vinogradovaa, Galina G. Malaninaa, Anireddy S. N. Reddyb, and Robert J. Flettericka,1
aDepartment of Biochemistry/Biophysics, University of California, 600 16th Street GH S412E, San Francisco, CA 94107; andbDepartment of Biology, Program
in Molecular Plant Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523
Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved March 30, 2009 (received for review November 3, 2008)
Much of the transport, tension, and movement in mitosis depends
on kinesins, the ATP-powered microtubule-based motors. We re-
port the crystal structure of a kinesin complex, the mitotic kinesin
KCBP bound to its principal regulator KIC. Shown to be a Ca2?
sensor, KIC works as an allosteric trap. Extensive intermolecular
interactions with KIC stabilize kinesin in its ADP-bound conforma-
tion. A critical component of the kinesin motile mechanism, called
the motor. KIC denies access of the motor to its track by steric
interference. Two major features of this regulation, allosteric
EF-hand ? motor ? calmodulin ? regulation
hydrolysis (1). Motor, neck, coiled-coil stalk, and tail domains
comprise a molecule of kinesin. The motor domain attaches
kinesin to microtubules and converts kinesin into an active
enzyme that hydrolyzes ATP for each step taken along the
microtubule. The tail domain anchors the specific kinesin’s
cargo, a vesicle, an organelle, a microtubule, or a multiprotein
complex (2). In most kinesins, a flexible coiled coil connects the
motor and tail domains and assembles kinesin into a dimer. The
conformational changes in the motor domains of kinesins that
lead to their ATP-powered directed movement along microtu-
bules are well described both structurally and biochemically
(3–5). A region of kinesin immediately before or after the motor
core, which is called the neck or neck linker, is a crucial element
for transmitting the conformational changes in the catalytic site
into the mechanical power stroke (6, 7). During the nucleotide
hydrolysis cycle, the neck linker adopts distinct positions with
respect to the motor domain, being either attached to the motor
core or released. In crystal structures, the N-terminal kinesins
are found with the neck linker docked along the motor domain
in the state reflecting their ATP-like conformation (8, 9). The
neck linker is observed to be detached when the N-terminal
kinesins are in the ADP-like state (10).
The regulation of kinesins is the least understood aspect of
their function. Cargo-binding proteins and proteins mediating
the binding of cargoes to the tail region of kinesins are presumed
to activate the kinesin motor (11, 12). Pausing of the motor
preventing unwanted activity depends on regulatory proteins or
internal interactions within the kinesin molecule (13–18). The
regulatory proteins modulating activity of the kinesins in re-
sponse to different cellular signals are still being identified. The
Ca2?ion has recently been shown to regulate Kinesin-1 via the
Ca2?-binding protein Miro interacting with kinesin through an
adaptor protein milton (18).
For the structural studies described in this article, we have
like calmodulin-binding protein) (19). KCBP is implicated in
formation of the bipolar spindles during nuclear envelope break-
down and the anaphase stage of mitosis by sliding and bundling
microtubules (20, 21). During metaphase and telophase, the
activity of KCBP is down-regulated to allow for greater micro-
olecular motors, kinesins, move along microtubules and
transport their cargoes by using the energy of ATP
tubules dynamics. The intracellular motor activity of this kinesin
during mitosis is presumably tuned by calcium signaling. In vitro,
its microtubule-stimulated ATPase activity and affinity to mi-
discovered specific regulator KIC (KCBP-interacting Ca2?-
binding protein) (22, 23). KIC requires 3-fold less concentration
of Ca2?(?1 ?M) than calmodulin to completely inhibit activity
of KCBP. Both KCBP and KIC are also required for trichrome
morphogenesis. Because the calcium signaling is thought to
occur through local gradients, regulation of KCBP by KIC and
calmodulin may be used differentially to produce a specific
response to an appropriate calcium concentration. KCBP con-
sists of the typical kinesin domains but, in addition, has a
specialized domain for binding regulatory proteins (24). Ca2?-
activated calmodulin or Ca2?-activated KIC binds to an ?-helix,
Through analysis of the 3-dimensional structure of KCBP in the
ATP-like conformation (25), the element preceding this helix
was termed the neck mimic because of its sequence and struc-
tural resemblance to the neck linker of the N-terminal kinesins
[supporting information (SI) Fig. S1]. This element is common
to all C-terminal kinesins. Docking of the neck mimic along the
motor core stabilizes the ATP-like conformation of the C-
terminal motor (25). In the ADP-bound conformation, the
interactions with the true neck, N-terminal to the motor core,
stabilize the motor core of C-terminal kinesins.
The binding of the regulator, calmodulin or KIC, disrupts
interactions of KCBP with microtubules. In the absence of
stimulation of its ATPase activity by microtubule binding, the
motor switches off (22, 23). Our goal was to discover the
regulation of this motor at high resolution and to possibly
elucidate the principles applicable to the regulation of other
Crystallization of KCBP-KIC Complex. In our crystallographic exper-
iments, we used Arabidopsis KCBP (amino acids 876-1261), a
monomer, and full-length Arabidopsis KIC. The domains and
their relation to the amino acid sequence are shown in Fig. 1A.
To crystallize the complex, the mutation C1131N was introduced
in the region of KCBP’s loop L11, which has variable confor-
mations in crystal structures of kinesins and often makes crystal
lattice contacts (26). The mutation did not affect microtubule
binding of KCBP nor its regulation by KIC. The recombinant
KCBP and KIC were separately expressed in Escherichia coli.
Author contributions: M.V.V. designed research; M.V.V. and G.G.M. performed research;
and M.V.V., A.S.N.R., and R.J.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The structure of KCBP-KIC complex have been deposited in the Protein
Data Bank, www.pdb.org (PDB ID code 3H4S).
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
www.pnas.org?cgi?doi?10.1073?pnas.0811131106 PNAS ?
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The complex of 2 proteins was isolated by using Ni2?-NTA
chromatography using a polyhistidine tag attached to KIC, later
removed by TEV-protease, and crystallized.
Crystal Structure of KCBP-KIC Complex. Fig. 1B shows the crystal
structure of KCBP-KIC complex refined to a resolution of 2.4 Å
(see Table S1 and Methods for data collection, refinement
statistics, and model building; see Fig. S2 for typical volumes of
the electron density map). The model encompasses most of the
sequence of the KCBP construct used for crystallization except
for a few residues belonging to the termini and to some surface
loops. Mg2?-ADP occupies the nucleotide pocket. A prominent
34-Å-long amphipathic helix that binds calmodulin or KIC is
observed between the neck mimic and a highly negatively
charged C-terminal coil of kinesin. The neck mimic, calmodu-
lin-binding helix, and negative coil comprise the 3-unit regula-
tory domain of KCBP (25) (Fig. 1 A and B).
A mostly helical protein, KIC wraps around the calmodulin-
binding helix of KCBP. The structural model of KIC (Fig. 1C)
was built de novo and comprises most of its sequence. The model
is missing the N-terminal segment preceding Asp-34 that is rich
in hydrophobic residues. Residues 19–22 (ETKY) were modeled
with confidence into the electron density map where the side
chains of Thr-20 and Tyr-22 contribute to the sites of hydro-
The 2 complex components make extensive contact (1,900 Å2
are buried in the interface), with numerous specific interactions
holding the 2 proteins together (identified below). Both the large
size of the interface and the specificity of the interprotein
interactions suggest that the observed conformation of the
complex is physiologically relevant and is not influenced by
crystal packing forces.
KIC Is a Previously Uncharacterized Ca2?Sensor. Sequence analysis
of the recently discovered KIC defined a group of Ca2?ion-
binding proteins featuring a single EF-hand motif (23). Surpris-
ingly, our structure revealed 2 EF-hands in KIC, one with a
canonical Ca2?-binding loop (Fig. S2B) (27) (helices C and D in
Fig. 1D) and another of nearly identical conformation (helices A
and B in Fig. 1D). However, because of its amino acid compo-
sition, the loop of this EF-hand will not bind metal ions (Table
S2). Both the Ca2?-independent EF-hand and the classical
usually described as ‘‘open’’ and observed for EF-hand pairs of
Ca2?-binding proteins [fast skeletal troponin C (28), calmodulin
(29)] loaded with Ca2?ions (Fig. S3). The familiar short
antiparallel ?-sheet (27) (Fig. 1D) stabilizing EF-hands pairing
is formed. The hydrophobic interactions between the helices of
the EF-hands further support the open conformation of the
EF-hands in KIC. An extensive surface-exposed hydrophobic
pocket consistent with the open conformation of the EF-hands
embraces the calmodulin-binding helix of KCBP. Remarkably,
the 3-dimensional organization of KIC reminds one of troponin
C without its C-terminal domain (Fig. S3 B and E). Even the
components. The fragments of KCBP and KIC visualized in the crystal structure are traced in orange for KCBP and in green for KIC. The domain structure of KCBP
is depicted. (B) Crystal structure of the complex of kinesin KCBP and KIC solved at a resolution of 2.4 Å. The motor (light orange) and KIC (green) are shown
schematically. ADP (light blue) and Ca2?ion (black) are space-filling models. The regulatory domain of KCBP is highlighted in orange. (C and D) Two views of
the structure of KIC in the complex. In C, KIC is shown in orientation allowing visualization of the part corresponding to the central helix of the Ca2?sensors
(calmodulin, troponin C). The C-terminal part of the ‘‘central’’ helix in KIC is less ordered than its N-terminal half. For alignment of KIC with troponin C and
the EF-hand pair is shown in cyan.
www.pnas.org?cgi?doi?10.1073?pnas.0811131106Vinogradova et al.
segment corresponding to the troponin C central helix is present
in KIC (Fig. 1C). The superposed positions of ?-carbon atoms of
KIC (amino acids 35–107) and calmodulin (amino acids 5–76)
(PDB ID code 1mxe) (30), or KIC (amino acids 34–111) and
troponin C (amino acids 13–89) (PDB ID code 1ytz) (28) show
RMS deviations of 1.73 and 2.03 Å, respectively. Thus, KIC,
troponin C, and calmodulin are structural relatives and belong
to the same group of EF-hand proteins, namely the subfamily of
Specific Interactions Between KCBP and KIC Define 2 Extensive Inter-
action Sites. There are 2 major sites of interactions between
KCBP and KIC. One was anticipated from the secondary
structure predictions of KCBP and biochemical experiments;
this is the classical target-recognition mechanism used by Ca2?-
binding proteins. The amphipathic calmodulin-binding helix of
KCBP is localized in the hydrophobic pocket of KIC. In the heart
of the hydrophobic pocket, Trp-1233 of calmodulin-binding helix
of KCBP is found (Fig. 2). The large hydrophobic side chain of
this residue coordinates ?9 residues of KIC, whereas the side
chains of other hydrophobic residues of KCBP (Leu-1226,
Leu-1229, Val-1230, Tyr-1232) make further complementary
hydrophobic contacts. These interactions define the position and
orientation of the calmodulin-binding helix inside KIC.
The calmodulin-binding helix, placed within the pocket in KIC
is only part of the extensive interface between the motor and its
regulator. The 2 proteins interact through a large surface area
with less than half (900 Å2) because of interaction via the binding
helix. The further contacts are forged between residues of the
KCBP neck mimic as well as the part of the kinesin core called
the ?-domain (32) (indicated in Fig. 1B) with the surface of KIC
including the loops of the EF-hands (Fig. 3). These contacts with
the surface of KIC, opposite from the traditional target-binding
site in the Ca2?-sensors, contribute another 1,000 Å2into the
complex interface and comprise the second interaction site.
In the second interaction site, there are 2 key clusters of
hydrophobic side chains. The side chain of KCBP’s Ile-1210
forms hydrophobic interactions with 3 side chains of KIC
interactions is formed around Ile-933 of KCBP, a residue
conserved in all KCBPs. This residue interacts with 4 hydro-
phobic residues of KIC including 2 located in the Ca2?-binding
loop region, Leu-88 and Thr-97. The side chain of His-55 located
in the Ca2?-independent EF-hand loop interacts with KCBP
residues Ala-1152 and Ser-1154 at the C-terminal part of its helix
?4; the latter is part of the switch II cluster of kinesins. Hydrogen
bonds also contribute to this interaction site; one links the main
chain of Ile-1210 and the side chain of Gln 96 of KIC, and
another is formed with the side chains of Asn 1212 of KCBP and
Lys-44 of KIC. The described interactions found in the second
site of the KCBP-KIC interface define the position and orien-
tation of KIC with respect to KCBP in the complex.
Conformational State of Kinesin. The observed conformation of
kinesin in the complex is clearly different from those determined
previously without KIC (Fig. 4A) (25, 26). Specifically, the
regulatory domain of KCBP is dislodged from the motor surface
and is configured differently. The neck mimic is unzipped from
its docked position along the motor core and now interacts with
KIC (Fig. 3). The calmodulin-binding helix grows longer by 4
helical turns within the KIC hydrophobic pocket. ADP occupies
the nucleotide-binding pocket in all KCBP structures, but the
positions of the switch II cluster helices (33) differ, representing
the ADP-bound conformation of the motor in the complex and
the ATP-like conformation in free KCBP (Fig. 4A). In the
absence of microtubules, these conformational states of kinesins
are of nearly equal free energy (9). Changes in buffer conditions
will favor one state over the other, resulting in different con-
formations observed in the crystal structures.
The ADP-bound conformation of KCBP differs significantly
from its ATP-like conformation not only by positions of the
nucleotide-sensing elements (switch I and switch II) but also by
positions of the whole regulatory domain including the neck
bic pocket of KIC. KCBP is in orange; KIC is in green. The selected interacting
residues are shown as stick models and specified. The calmodulin-binding
helix is shown as a ribbon. The secondary structure elements of KIC are shown
Interactions of calmodulin-binding helix of KCBP with the hydropho-
and between the EF-hands loops of KIC and KCBP. KCBP is in orange; KIC is in
green. Ca2?is shown as a black sphere. The secondary structure elements of
both proteins are shown schematically and labeled as in Fig. 1. Principal
better figure presentation, we chose not to show Gln96 of KIC.
Interactions formed between the residues of the neck mimic and KIC
Vinogradova et al.PNAS ?
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KIC Recognizes ADP-Bound Conformation of KCBP. KIC binds tightly
to residues of KCBP that are exposed in the ADP-bound state
but would not be available in the ATP state. Upon hydrolysis of
ATP, helix ?4 of switch II moves toward the tip of the motor core
(Fig. 4A). Influenced by the movement of helix ?4, the hydro-
phobic pocket on the surface of kinesin shifts and exchanges
Ile-1210 at the base of the neck mimic (Ile-1210 is conserved in
kinesins and positioned like Ile-325 in Kinesin-1) to Ile-890 of
the motor ?1 strand (Fig. 4 B and C). The liberated Ile-1210 dips
into a hydrophobic cavity provided by KIC (Fig. 3). The released
neck mimic is fixed to the surface of KIC by several comple-
mentary contacts including electrostatic and hydrophobic side-
chain interactions (Fig. 3). Additionally, because helix ?4 is
moved, His-55 of KIC is accommodated between Ala-1152 and
Ser-1154 at the C-terminal part of the helix ?4. Thus, binding of
KIC stabilizes the ADP-bound conformation of the motor.
Modeling of Complex on Microtubules.WemodeledtheKCBP-KIC
structure on microtubules (Fig. S4) to investigate how binding of
KIC affects the relationships of this kinesin with microtubules.
The model was built by superposition of the KCBP structure in
a complex with KIC and the structure of KAR3, a Kinesin-14
motor, bound to microtubules in ADP state (34). KIC clashes
with tubulin, and this fact is sufficient to promote the dissocia-
tion of KCBP from microtubules. In addition, the calmodulin-
segment after the helix is pointed toward the negatively charged
C terminus of tubulin. The repelling electrostatic interactions
between this C-terminal region of KCBP and microtubules
surface might contribute to the destabilization of the KCBP–
microtubules complex in the presence of KIC. However, the
effect of the charge repulsion as a part of regulatory mechanism
of KCBP seems not to be crucial, because the deletion of the
negative coil does not affect the regulation of KCBP by KIC.
Many biochemical and functional studies of this complex were
recently published (23). We determined and analyzed the crystal
structure of kinesin motor KCBP in complex with its physiolog-
ical regulator KIC, a previously uncharacterized EF-hand Ca2?
sensor. The recognition site formed between KCBP and its
regulator extends beyond the motor’s special binding domain to
engage complementary surfaces of 2 proteins. Our results show
its regulator is tuned in response to specific signaling, first of all,
by binding of the Ca2?ion and subsequent conformational
changes modifying the entire surface of the Ca2?sensor. Upon
binding of a single Ca2?ion, the surface of the EF-hand Ca2?
sensor undergoes significant changes that have 2 consequences,
the first being the opening of the hydrophobic pocket for binding
the specialized helical motif of its target. The second, previously
unappreciated, consequence is that the surface of the EF-hands
loops, transformed by Ca2?ion binding, is an equal player in
the target-recognition mechanism. This surface is designed to fit
the particular conformation of its motor target and makes the
regulation highly specific.
The conformation of kinesin in the complex, producing the
matching surface for interaction with KIC, is ADP bound.
Consistent with the proposed role of imitating the properties
of the N-terminal kinesin neck (7, 25), a critical component of
motility, the neck mimic stabilizes the ATP conformation of
KCBP by being zipped along the motor. This element was
predicted to become free when the motor conformation changes
to the ADP-bound. The dislocation of the regulatory domain in
the ADP state of KCBP is the direct result of the conformational
transformation of the motor reflecting its nucleotide state.
Indeed, in the ADP state of KCBP, the neck mimic is unbound
from the motor core. Therefore, the structure is important
evidence of the nucleotide dependent transformations of the
neck mimic, supporting the assigned role of this element in the
kinesin catalytic cycle.
As a result of the changed motor conformation, many residues
become exposed and available for interactions with KIC. These
mutually stabilizing, complementary interactions between the
motor and its regulator form the extensive specific interface and
unambiguously define the relative orientation of the complex
components. In the observed conformation of the complex, the
neck mimic is found sequestered and, therefore, disengaged
from further participation in the conformational events associ-
ated with nucleotide hydrolysis and related mechanical cycles of
In this work, the previously proposed mechanism of KCBP
regulation (25) is elaborated (Fig. S5). The essential strategy for
this regulation is that binding of Ca2?-activated KIC stabilizes
the motor in the ADP state or in a similar conformation in the
absence of nucleotide. KIC disallows ATP hydrolysis by KCBP
by fixing the position of the neck mimic on its surface. The
immobilized neck mimic is no longer available for interactions
with the motor core that are required for achieving a stable
conformation when ATP binds. The motor is arrested in a state
characterized by weak affinity for microtubules. The interactions
of KCBP with microtubules are destabilized further by steric
hindrance between KIC and tubulin and electrostatic repulsion
between the negatively charged clusters in the C-terminal re-
gions of KCBP and tubulin.
(A) The structure of KCBP-KIC complex is superposed with the structure of
KCBP alone (PDB ID code 3cob, chain A). Only the switch II helix ?4 and the
regulatory elements (in pink) of the solo KCBP structure are shown. The
analogous elements of KCBP in the structure of the complex are highlighted
in orange. For our previous crystallographic studies of KCBP (PDB ID codes
1sdm, 3cob, 3cnz), we used KCBP from potato (amino acids 884-1252) that is
80% identical to Arabidopsis KCBP. (B) The hydrophobic pocket (marked in
yellow) on the kinesin surface (gray) is occupied by Ile-1210 in the ATP-like
conformation. (C) The shifted hydrophobic pocket (marked in yellow) on the
kinesin surface (gray) is occupied by Ile-890 of the ?1 strand as found in the
ADP state. Ile-1210 is expelled from the hydrophobic pocket on the kinesin
surface and interacts with KIC (Fig. 3). Hydrophobic residues are conserved at
positions 890 and 1210 in all kinesins. Helices ?4 and ?6 and the ?1 strand of
KCBP (indicated) are shown schematically and are visible through the trans-
lucent surface. The neck mimic is shown as a coil and is colored according to
the conformational state.
The conformational change in KCBP accompanying ATP hydrolysis.
www.pnas.org?cgi?doi?10.1073?pnas.0811131106Vinogradova et al.
The native KCBP may form a dimer like some other Kine-
sins-14 (6, 22). Binding of KIC takes place, likely within the
proximity of the second motor head, and so may influence its
orientation and, therefore, the behavior of the dimer. However,
the relevance of this suggestion needs further experimental
justification and cannot be warranted based on available data.
The mechanism suggested by findings presented in this article
advance our knowledge of kinesin regulation. Our work shows
that one strategy to stop a kinesin motor is to trigger its
association with a regulatory protein that interferes with the
catalytic or mechanical cycles of kinesin by blocking nucleotide-
responsive elements of the motor. Given the structural similarity
and the analogous roles in nucleotide-induced conformational
kinesins and the neck mimics in minus-end kinesins could be
targeted by regulation in an analogous way. Sequestering the
neck linker or its mimic would interrupt the nucleotide cycle
even in the presence of ATP and microtubules and cause the
motor to pause. In addition, similar to the regulation of KCBP
by KIC, steric hindrance of microtubule binding is likely to be
exploited in the regulation of other kinesins.
Protein Expression and Complex Isolation. Arabidopsis KIC was subcloned into
a modified pRSFduet plasmid (Novagen) adapted for Gateway cloning tech-
nology (Invitrogen). The plasmid encoded the N-terminal His6tag separated
from the expression gene by a linker with the TEV-protease cleavage site.
Arabidopsis KCBP (amino acids 876-1261) was cloned into pET28b vector
(Novagen) by using NcoI-EcoRI sites carrying no tag. Mutation C1131N was
introduced into cDNA of KCBP according to a protocol and by using the
reagents of the QuikChange site-directed mutagenesis kit (Stratagene). For
protein expression, these constructs were transformed into E. coli-competent
harvested. The cell pellets containing the recombinant KCBP and KIC were
combined and subjected to lysis by sonication in the buffer containing 50 mM
Tris (pH7.5), 50 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.1 mM ATP, 1 mM TCEP,
and protease inhibitors mixture. KCBP was provided in excess to ensure that
all KIC is bound in a complex. The soluble fraction of the lysate was loaded on
the Ni-NTA beads (Amersham). The bound KCBP-KIC complex was eluted in
the presence of 100 mM imidazole. The complex was treated with TEV-
was passed through the Ni-NTA beads again. The unbound fraction contain-
ing the tag-free KCBP-KIC complex was collected. Before crystallization, the
complex sample was concentrated up to 15 mg/mL.
Crystallization and Data Collection. Crystals were grown by using the vapor-
diffusion method, in sitting drops under the following conditions: 30% PEG
crystals were frozen in liquid nitrogen. Data collection was done at the
Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley,
CA) Beamline 8.3.1 (? ? 1.1 Å) by using a single crystal. Data were integrated
by using DENZO and scaled with SCALEPACK. The crystal of the complex was
of the hexagonal space group P6522 with cell dimensions a ? b ? 118.8 Å and
c ? 142.1 Å. The asymmetric unit contained 1 molecule of the complex with
molar ratio of KCBP/KIC of 1:1 (57 kDa). The solvent fraction of the crystal is
49.7%, and the Matthews coefficient is 2.5 A3/Da.
Molecular Replacement and Model Building. The structure of the complex was
determined by the molecular-replacement method using the CNS algorithms
with a starting model derived from the atomic coordinates for residues
coefficients 2Fo-Fc were calculated from the phases of the initial model.
Subsequent rounds of model building and refinement were performed by
using the programs Coot (CCP4 Package) and CNS, respectively. The electron
density corresponding to the regulatory protein (KIC) was visualized at early
stages of model building. The crystallographic model is refined to 2.4 Å, with
R/Rfreevalues of 22.3/22.8, and contains the polypeptide chains of KCBP (359
aa) and KIC (96 aa), 160 water molecules, 1 Mg2?–ADP complex, 1 Mg2?ion
N-terminal amino acid residues of KCBP (amino acids 876–879 and 881–885),
although Tyr-880 was modeled into electron density. There was no visible
electron density for loop regions of KCBP (amino acids 1030–1034 and amino
acids 1075–1083), and the C-terminal region (amino acids 1256–1261). Only 4
acids 126–131) remained invisible. See also Table S1 for more details on data
collection and refinement statistics.
KAR3 bound on microtubules. This work was supported by National Institutes
of Health Grant P01 AR42895 (to Roger Cooke) and National Science Foun-
dation Grant MCB-0079938 (to A.S.N.R.).
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