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 ?
May 19, 2009 ?
vol. 106 ?
no. 20 ?