34
Structures of kinesin and kinesin–microtubule interactions
Eckhard Mandelkow* and Andreas Hoenger†
Several X-ray crystal structures of kinesin motor domains
have recently been solved at high resolution (∼0.2–0.3 nm),
in both their monomeric and dimeric states. They show the
folding of the polypeptide chain and different arrangements
of subunits in the dimer. In addition, cryo-electron
microscopy and image reconstruction have revealed
microtubules decorated with kinesin at intermediate
resolution (∼2 nm), showing the distribution and orientation
of kinesin heads on the microtubule surface. The comparison
of the X-ray and electron microscopy results yields a model
of how monomeric motor domains bind to the microtubule
but the binding of dimeric motors, their stoichiometry, or the
influence of nucleotides remains a matter of debate.
Addresses
*Max-Planck-Unit for Structural Molecular Biology, Notkestrasse 85,
D-22607 Hamburg, Germany; e-mail: [email protected]
†European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012
Heidelberg, Germany; e-mail: [email protected]
Current Opinion in Cell Biology 1999, 11:34–44
http://biomednet.com/elecref/0955067401100034
© Elsevier Science Ltd ISSN 0955-0674
Introduction
Kinesins have diverse functions such as the transport of
vesicles along microtubules and the segregation of chromosomes in mitosis. Correspondingly, many variants of
kinesins have been identified. Recent reviews have covered different aspects of kinesin: intracellular transport
[1–3], mitosis and meiosis [4–6], biochemistry and kinetics
[7–9], micromechanics [10,11], subunit composition
[12,13], and evolution [14,15]. In addition, the crystal
structures of kinesin and their relationship with myosin
and G proteins have been surveyed [16–18]. In the past
2–3 years several groups have studied the structure of
microtubules decorated with kinesin motor domains by
electron and cryo-electron microscopy and image reconstruction [19–22,23••,24•,25•,26–29]. This approach has
gained added weight with the recent X-ray crystal structures of monomeric and dimeric kinesin-like motors
[30,31,32••,33•,34••,35•] and with the solution of the structure of tubulin by electron crystallography [36••,37•]. The
results open the prospect of understanding the
kinesin–microtubule interaction at high resolution. We
focus our review on the relationship between the crystal
structures of kinesins and the image reconstructions of
decorated microtubules, and the implications of these
results for microtubule-based motility.
X-ray crystal structures of kinesins
Different kinesins can be subdivided on the basis of their
directionality (moving towards the plus or minus end of
microtubules), the position of the motor domain (near the
amino terminus, in the middle, or near the carboxyl terminus of the polypeptide chain, that is N-, M-, or C-type
kinesins), or by their subunit composition (monomers,
homodimers, heterodimers, heterotrimers, heterotetramers, combinations with light chains, and so on). The
common feature is the ~40 kDa globular motor domain
which generates force and binds ATP and microtubules; in
addition most kinesins have an α-helical stalk domain acting as a spacer and force transducer, and a globular tail
domain that binds the light chains and connects the complex to the cargo (e.g. vesicles, organelles, chromosomes).
There are six published X-ray studies of recombinant
kinesin motor domains (four in monomeric states, two in
dimeric states), which provide eight independent views of
the structure: the ‘conventional’ kinesin (a plus-end directed N-type kinesin) in the monomeric form from humans
([30]; Brookhaven Protein Data Bank code 1bg2), conventional kinesin from rat in the monomeric and dimeric form
([33•,34••]; Brookhaven Protein Data Bank codes 2kin and
3kin], the minus-end directed C-type motor Ncd from
Drosophila in the monomeric and dimeric form ([31,32••],
Brookhaven Protein Data Bank codes 2ncd and r2ncdsf),
and the minus-end directed C-type motor Kar3 from yeast
in the monomeric form ([35•]; 3Kar). These motor domains
are similar in their structure; they consist of a central
β sheet of eight strands, sandwiched between six α helices,
three on either side (Figure 1). The ATP binding site lies
at the front face of the motor domain, while the putative
microtubule-interacting region determined by mutagenesis (alanine screening) and limited proteolysis ([38••,39•];
shown in green on kinesin in Figures 1 and 3) lies on the
rear surface.
The motor domain can be subdivided into several functional regions, the ‘core’ motor domain (strand β1 up to the
end of helix α6, residues 10–324 in conventional kinesin
from rat brain), the linker regions forming the entry and
exit to the core motor (amino-terminal, strand β0, residues
1–9; and carboxy-terminal, strands β9 and β10, residues
325–338), and the neck helix α7 which forms the initial
part of the stalk (residues 339–370). The amino- and carboxy-terminal linker regions are closely adjacent in the
three-dimensional structure so that the motor domain can
be suspended anywhere in the amino-acid chain
(Figure 1a), thus generating N-, C-, or M-type motors. The
linker regions appear to be important for the direction of
movement [32••,40••,41•,42••].
The neck helix of kinesin (beginning at residue 339 in the
conventional kinesin sequence from rat) forms a coiled-coil
interaction if the helix is sufficiently long which leads to
dimerization. This occurs not only in the crystal, but also in
solution [43,44•,45,46•]. The dimerization is achieved by
Structures of kinesin and kinesin–microtubule interactions Mandelkow and Hoenger
35
Figure 1
(a)
Rat brain
kinesin
(b)
Drosophila
Ncd
N
C
N
α5
+
C
Microtubule
axis
α4
(c)
(i)
(ii)
rK379 (AMP-PNP)
(iii)
rK379 (no nucleotide)
Dimeric Ncd [25•]
Current Opinion in Cell Biology
(a) Structure of dimeric rat brain kinesin (Kozielski et al., [34••]) (b) Structure of dimeric Drosophila Ncd (Sablin et al. [32••]). (a) The subunit of
kinesin in bold colors shows a core β-sheet (blue) with surrounding α-helices (red). In this orientation, the nucleotide binding site is near the bottom
(ADP, gold), the carboxy-terminal neck helix (red) projects at the upper end to the right, the presumptive microtubule binding region (green) is at the
rear surface. The neck helix of one monomer (red, starting at residue 339) forms a coiled-coil with the neck helix (gray) of the second monomer.
Note that the two heads are related by a 120° rotation about an axis close to that of the coiled-coil. (b) Ncd dimer, shown with the bold coloured
subunit in the same orientation as kinesin in (a). In this case, the linker and neck helix (red) protrude from the amino-terminus. The second Ncd head
forms a coiled-coil interaction with the neck helix of the first head so that the two Ncd subunits are related by a 180° rotation. The diagram
illustrates that the core motor domains of kinesin and Ncd are similar, but the positions of the neck helices and the arrangement of the dimers are
very different. Neither of the crystal dimer structures matches equivalent binding sites on the microtubule lattice. In both cases the bold subunit is
orientated as if it were bound to a microtubule behind it, with the plus-end pointing up (so that the green region interacts with tubulin); see [23••]
and (c). (c) Image reconstructions of microtubules decorated with (i) the dimeric kinesin construct rK379 in the presence of the nonhydrolysable
analogue of ATP AMP-PNP, (ii) dimeric kinesin construct rK379 without nucleotide, (iii) dimeric Ncd-450 with AMP-PNP. Note that kinesin attaches
to the microtubule with only one head per tubulin heterodimer in all cases (stoichiometry =1), while Ncd attaches with two heads
(stoichiometry = 2). The position of the tightly bound head is indistinguishable within the error of the method in all cases, and there are no marked
changes with different nucleotides (AMP-PNP; ADP [not shown], or no nucleotide). Clear differences in the outer shape of the tightly bound heads
are also observed between kinesin (i, ii) and Ncd (iii).
36
Cytoskeleton
Figure 2
(a)
(b)
(c)
(d)
rK379 (AMP-PNP)
(e)
(a) Cryo-electron micrograph of a
15-protofilament microtubule decorated with
dimeric rat kinesin construct rK379.
(residues 1–379) (b) Typical diffraction
pattern of such microtubules. (c) Averaged
and reconstructed decorated microtubule.
Note the dense labeling with kinesin heads,
with an axial periodicity of 8 nm,
corresponding to the spacing of a tubulin
dimer. (d–f) Axial projections of microtubules
(viewed from the plus-end to the minus-end)
decorated with (d) dimeric rat kinesin (rK379)
in the presence of AMP-PNP, and (e) without
nucleotide. The motors are visible as little
protrusions which slew slightly in an
anticlockwise direction. The observed mass
corresponds to one motor per tubulin dimer.
(f) When compared to microtubules
decorated with a dimeric Ncd construct
(residues 450–700), it becomes obvious that
in the case of Ncd two motors are bound per
tubulin dimer (additional mass at higher radii).
rK379 (no nucleotide)
(f)
Helical image reconstruction
Dimeric Ncd
Current Opinion in Cell Biology
the second half of the neck helix (residues 355–370) which
shows a leucine-zipper-like interaction [47], whereas the
first half of the neck helix (residues 339–355) contains nonstandard hydrophilic and charged residues that weaken the
coiled-coil interaction and may allow the two heads to split
apart, as required in hand-over-hand motility models (see
below). The two neck helices of kinesin have the two-fold
symmetry expected of a coiled-coil (axis in the plane of the
paper in Figure 1b), but the heads are related by a ~120°
rotation (rotation axis close to that of the coiled-coil axis), a
conformation that is not compatible with an equivalent
binding of each head to microtubules. The crystal structure
of the kinesin dimer is probably close to that occurring in
solution [48], but when kinesin binds to a microtubule the
two heads splay apart and bind to different tubulin subunits
[23••,46•]. This can be achieved by the separation of the
neck helices and/or a rearrangement of the linker region
([23••,49•]; see Figure 3bi).
In the case of the Ncd dimer structure [31], both core motor
domains (extending from β1 to α6 in each chain) are similar to that of the Ncd monomer or of kinesin (Figure 1);
however, the linker sequence enters from the amino-terminal side and adopts a mostly α-helical conformation which
replaces the strand β0 of kinesin before it continues into
the core domain. In contrast to kinesin, the two motor
domains of Ncd show the same two-fold symmetry as their
amino-terminal coiled-coil neck domains; therefore the two
dimeric motor complexes have very different quaternary
structures, but in either case they would have to undergo a
major change in conformation in order to achieve an equivalent binding of both heads to microtubules.
Structures of kinesin and kinesin–microtubule interactions Mandelkow and Hoenger
37
Figure 3
(a) Docking of the crystal structure of dimeric
kinesin or Ncd into the density of decorated
microtubules obtained by image reconstruction.
(i) (ii) Kinesin dimer crystal structure [34••]
docked into the reconstructed electron density
of kinesin-decorated microtubules [23••]. One
of the two kinesin heads (bold colors)
completely accounts for the observed density,
the second head (grey) lies outside the density
contours; this indicates that the arrangement of
the dimeric kinesin heads is disrupted when
binding to the microtubule. In (i), the bound
head has the same orientation as the bold head
in Figure 1a, in (ii) the microtubule is seen endon from the plus-end to the minus-end (the
inside of the microtubule is at the upper edge).
(iii, iv) Ncd dimer crystal structure [32••]
docked into the reconstructed electron density
of Ncd-decorated microtubules [25•]. In this
case, the density includes a volume equivalent
to two motor domains. The motor domain
directly associated with microtubules (bold
colors) has the same orientation as the bold
head in Figure 1b and the bold head in ai. The
second head of the Ncd crystal structure is
partly outside the density, indicating that it
undergoes a rearrangement when the Ncd
dimer binds to the microtubule. Although the
second head can be translated and rotated to
fit into the density contours, the orientation is
not known with certainty (one possibility is
given in [25•]). (b) Modeled structural
conformations of the kinesin dimer during
interaction with microtubules. (i) The X-ray
structure of the kinesin dimer, with the lower
head docked into the density of decorated
microtubules [23••]. Whereas the position of
head B (bold colors) was confirmed by electron
microscopy, the second head (grey) shifts its
position, probably to the next free binding site
in the plus-end direction (transparent model).
(ii) One way to span the resulting 8 nm
distance between two bound motors is that the
coiled-coil opens substantially. (iii) Another
possibility is that the kinesin linker region
(yellow) is capable of flipping backwards on the
leading head which would require a less
extensive opening of the neck helix [49•].
(a)
(ii)
(i) Rat kinesin
(iv)
(iii) Drosophila Ncd
(b)
(i)
(ii)
(iii)
Current Opinion in Cell Biology
Kinesin binds nucleotides which are embedded in four
contact regions (yellow in Figure 1) that share remarkable
structural homologies with G proteins and myosins [30,50].
The nucleotide-binding regions have been termed
N1–N4, by analogy with G1–G4 of p21ras. N2 and N3 correspond to the switch regions Switch I and Switch II of
p21ras; they relay conformational changes during
nucleotide turnover to the rest of the molecule. In the case
38
Cytoskeleton
of G proteins and myosin, the conformations have been
determined in different nucleotide states [51–53], whereas
the kinesin structures known thus far contain bound ADP,
revealing only one conformation around the nucleotide
binding pocket. This conformation is unusual, because it
shows a tight closure of the nucleotide binding cleft which
in G proteins and myosin is typical for the ATP bound
state but not for the more open ADP bound state [18];
these features may be related to the differences in the
binding and hydrolysis of nucleotides [9].
Image reconstructions of microtubules
decorated with kinesins
Microtubules can be decorated with kinesin in a periodic
fashion such that one kinesin ‘unit’ binds to every αβtubulin heterodimer, with a preferential interaction
between β tubulin and kinesin [54]. This accentuates the
dimer lattice of the microtubules and shows that it is of
the ‘B’-type, with adjacent protofilaments staggered by
~0.9 nm (Figure 2). The decoration shows intrinsic polarity and can therefore be followed to the end of
microtubules revealing the polarity of the decorated
microtubules [55–57]. The plus-end has a crown of βtubulin subunits, the minus-end terminates with a base of
α-tubulin subunits, consistent with subunit-specific antibody labeling [58] and the position of the exchangeable
GTP on β-tubulin at the plus end [59]. These features of
microtubules had been a matter of debate for some time
but now appear to be generally accepted. The high resolution structure of tubulin indeed reveals that the
exchangeable GTP is located on the exposed surface of β
tubulin [36••].
The bound kinesin adopts the symmetry of the microtubule lattice and therefore this aggregate lends itself to
electron microscopy combined with image reconstruction
(Figures 1b and 2). For frozen-hydrated and unstained
microtubules one can obtain resolutions of ~2 nm, which
is sufficient to deduce the positions and molecular
envelopes of the tubulin and kinesin subunits, but not the
folding of the polypeptide chain or the atomic coordinates. In contrast to X-ray crystal structures which can be
refined against precisely known bond angles and distances, no such refinement is possible for electron
microscopy/image reconstruction structures. The results
are therefore more dependent on experimental procedures and interpretation which may vary between
laboratories. This probably explains why, despite considerable efforts, different groups have come to conclusions
which are at variance with one another, in contrast to the
X-ray crystal structures which are highly reproducible
between laboratories.
Image reconstructions of decorated microtubules were
initially obtained with monomeric kinesin or Ncd constructs [19,22,29] and later extended and confirmed
[20,21,23••,24•,25•,26,27]. There is a broad agreement
that the motor domain is located roughly at the crest of
the protofilament, somewhat displaced in a counterclockwise direction when the microtubule is viewed with its
plus-end up (this asymmetry allows the absolute orientation of decorated microtubules). Monomeric kinesin and
Ncd are very similar, and share the same binding site on
microtubules, consistent with biochemical evidence
[60,61]. Beyond this, the published results diverge on
several issues.
Conformational changes induced by nucleotide binding
Two groups have reported substantial differences in conformation between motors bound to ATP analogues, ADP,
or no nucleotide [19,21,27], whereas two other groups
(including our findings) report that these structures are
similar within the error of the method [23••,25•]. These
changes are mostly observed in the second (loosely bound)
head, but not in the first (tightly bound) one. In this context it should be noted that the nucleotide content of the
bound kinesin is not precisely known but is probably low
as the nucleotide is released upon binding of the motor
domain to the microtubule [62,63•,64]. The reported conformational changes differ between kinesin and Ncd and
are therefore used to reinforce models of why these motors
move in opposite directions. Given the discrepancies
between the results the issue is likely to remain controversial for some time.
Arrangement of kinesin or Ncd dimers on the
microtubule surface and stoichiometry of binding
Kinesin and Ncd normally form homodimers in solution
(if the coiled-coil neck helix is present), and the question arises of how the two heads are accommodated on
the microtubule surface. The consensus is that the first,
tightly bound head binds in a similar way to the
monomeric motor domain described above, leaving the
arrangement of the second, loosely bound (or ‘tethered’)
head open. Here one has to distinguish between kinesin
and Ncd. In the case of Ncd there is agreement that
both heads bind on top of one β-tubulin subunit (one
directly and tightly, the other loosely tethered to the
first head). This yields a stoichiometry of 2, i.e. two
motor heads per tubulin heterodimer [20,25 • ,26].
Indeed, the cryo-electron microscopy data show an
attached mass equivalent to ~2 motor domains at roughly equal heights on the protofilament.
In the case of kinesin dimers there are two divergent
views. Two groups presented an arrangement that places
the second head slightly above the first head, tilted in a
counterclockwise direction when the microtubule plus
end is up [20,26]. The second head is visible only with
a small fraction of its expected volume. The authors
interpret this to mean that the second head is disordered, making it mostly invisible to helical image
reconstruction methods (yet the stoichiometry would
still be 2 for this model, as for Ncd). By contrast, we and
others find that there is only one bound motor head per
tubulin heterodimer, arguing that the two heads of the
Structures of kinesin and kinesin–microtubule interactions Mandelkow and Hoenger
kinesin dimer come apart upon binding to the microtubule and attach to two different β-tubulin subunits,
yielding an effective stoichiometry of 1 ([23 • ];
Milligan RA, personal communication]. This view is
supported not only by the cryo-electron microscopy
data, but also by measurements of the stoichiometry
using biochemical assays and mass determination by
scanning transmission electron microscopy. In addition,
decoration of tubulin sheets with dimeric kinesin constructs sometimes yields a 16 nm periodicity consistent
with the pairing of kinesin heads on adjacent tubulin
heterodimers [46 •]. Note, however, that stoichiometries
can also deviate from integral values. This could be
explained by several mechanisms, such as nonspecific
attachment of motors to the microtubule, or attachment
with lattice defects.
Fit of the X-ray structure into the electron density of
cryo-electron microscopy
To obtain a molecular understanding of the microtubule–motor interaction it is necessary to fit the high
resolution structures of kinesin and tubulin into the low
resolution electron density maps of decorated microtubules. As a first step, several groups have docked the
crystal structures of kinesin or Ncd onto the electron
microscopy maps of microtubules [23••,25•,28]. The next
step is to fit the tubulin structure into the electron
microscopy map of microtubules [37•] as this will allow us
to refine the docking of the motor structures
(Milligan RA, Downing K, personal communication). In
all cases, the major problem is that the electron
microscopy density provides relatively weak constraints
for fitting the crystal structures, especially in the present
case where all subunits are globular to first approximation.
This makes it necessary to introduce other constraints
(e.g. from biochemical or mutagenesis experiments) and
personal judgment, and it is therefore not surprising that
there are considerable discrepancies between the docking
models, (as explained below).
The first (tightly bound) head
For monomeric kinesin or Ncd, as well as for the tightly
bound head of dimeric kinesin or Ncd, the docking models of Sosa et al., [25•] and Hoenger et al., [23••] are
shown in Figure 3. If the microtubule plus end is chosen
to point upwards, the motor heads are oriented with the
ATP binding site at the lower tip, and the neck helix at
the upper end (as in Figure 1). Thus the long axis of the
motor domain is roughly parallel to the microtubule axis,
and the neck helix of kinesin points tangentially to the
microtubule surface in a counterclockwise direction.
The bound kinesin and Ncd motor domains have essentially the same position and orientation on the
microtubule [24•]. An important additional constraint is
to allow the rear surface of the head, containing the
region α4–loop12–α5, to interact with the microtubule.
This motif contains highly conserved residues that are
important for microtubule binding and motility, as shown
39
by site-directed mutagenesis [38••], and the rear surface
becomes protected against limited proteolysis by interaction with the microtubule [39•]. The orientation of the
tightly bound head is different in other docking models,
partly because they assume that that second head is
attached to the first in the same orientation as in the
crystal structure [28]. The need to accommodate two
heads in spite of missing electron density (see above)
leads, in our opinion, to a poor overall fit and a clash with
biochemical constraints.
Second (loosely bound) head of kinesin
As mentioned above, we have to distinguish between
kinesin and Ncd. In the case of microtubules decorated
with dimeric kinesin constructs we find the same occupancies as with monomeric kinesin constructs, one head
per tubulin heterodimer (stoichiometry = 1), and with
the same electron densities. Therefore all motor heads
are bound in the same orientation as described before for
the monomer (ATP site at lower end, compare with
Figure 1). This raises an interesting problem. How can
the tightly associated heads of a kinesin dimer bridge the
gap between adjacent tubulin subunits, and how can the
two heads bind simultaneously, given that the crystal
structure shows two nonequivalent orientations? This
issue is part of the more general question of stepping
models (see below). The problem is solved by assuming
that the two heads splay apart and reorient when they
attach to the microtubule in the rigor state. The first half
of the neck helix contains nonstandard residues with
almost no coiled-coil potential [44•,45,47] which would
make the splaying of the coiled-coil plausible; furthermore, the linker region connecting the core motor
domain and the neck is only loosely attached to the side
of the central β sheet and could also reorient without difficulties to allow separation of the heads [49•]. Tubulin
sheets decorated with dimeric kinesin constructs often
show an additional axial periodicity of 16 nm [46•]; this
suggests that the two separated heads bind to successive
β-tubulin subunits along one protofilament. The remainder of the coiled-coil segments would generate a spacing
of 2 × 8 = 16 nm, as illustrated in Figure 3bi.
Second head of Ncd
In the case of Ncd the stoichiometry is 2 so that both heads
of an Ncd dimer are attached to a given tubulin heterodimer, and a docking model for both heads was
proposed [25•]. The subsequent X-ray structure of dimeric Ncd [32••] revealed that the packing of the two heads on
the microtubule must be different from that in the crystal
(Figure 3aii). Thus, dimeric kinesin and Ncd are comparable in that both undergo gross conformational changes
upon interaction with the microtubule, although these
changes take different forms in the two motors (which may
be relevant for their directionality). Although the electron
densities of the two bound Ncd heads are well defined
there are fewer constraints for the orientation of the second
head (e.g. no guidance from microtubule-binding loops),
40
Cytoskeleton
and therefore the docking of this head proposed [25•] may
not be the only solution.
Other constraints for motor–microtubule
interactions
As mentioned above, structural constraints alone are not
sufficiently reliable to determine the interactions
between motors and microtubules because of limitations
inherent in the methods. We therefore discuss some
recent results from other methods that have a bearing on
the structural interpretations
Kinetics
Detailed studies of the kinetics of nucleotide hydrolysis
and microtubule binding of kinesin and Ncd in solution
have
been
performed
by
several
authors
[7,43,62,63•,64–68]. Kinesin differs from Ncd (and the
analogous case of myosin) by its high degree of processivity which ensures that it stays bound to a microtubule over
long distances without dissociating. This is consistent
with a hand-over-hand model of kinesin ‘walking’ (see
below). The rate of the microtubule-activated ATPase is
typically ~20 per second per head for dimeric kinesin.
Velocities are ~0.5–1 µm per second for kinesin, they are
much slower for Ncd, ~ 0.01 µm per second [60], but can
also be much faster for some fungal kinesins, ~2–3 µm per
second [69]. One can derive models relating the biochemical steps and rate constants to the structural
rearrangements [9]; however, from a purely structural
point of view only two reference states are known (solution and microtubule-bound in rigor) so that models of
conformational transitions will remain speculative until
further data are available.
Single particle observation and micromechanics
Experiments with optical tweezers or mechanical sensors
have shown that the movement of conventional kinesin
takes place in 8 nm steps, that two heads are needed for
processive movement, and that 1 ATP is hydrolyzed per
8 nm step [70•,71•,72,73,74•]. Using evanescent wave
microscopy and fluorescently labeled molecules it is possible to observe the movement of single kinesin molecules
along microtubules, confirming directly the high processivity of kinesin, when compared with other
motors [49•,75].
Mutagenesis
Point mutations in kinesin and Ncd have been found or
introduced in order to address four questions — the mechanism of nucleotide hydrolysis, the interaction with
microtubules, the role of the neck and stalk region, and the
determinants of directionality.
Mutations in the ATP binding site have generally confirmed that the conserved motifs and surrounding regions
(phosphate-binding-loop, switch I and II) are important for
motor activity ([50,76]; E Taylor, personal communication).
So far, it is not clear how nucleotide binding and hydroly-
sis are converted into conformational changes. Simulated
annealing methods have been used for predictions, but
experimental confirmation is not yet available [77]. The
comparison with myosin or G proteins suggests that
kinesin may follow a different mechanism, since its
nucleotide-binding pocket is noncanonical, that is ‘tight’
instead of ‘open’ in the ADP bound state [18].
Mutations of exposed residues of kinesin have provided
strong evidence that the area around α4–loop12–α5 and
other loops on the ‘rear’ side of the molecule (shown in
green in Figure 1a) make the contact with microtubules
[38••], consistent with protease protection assays [39•].
This constraint is incorporated into the models of
Figures 1c and 3. Conversely, the interactions with tubulin have been probed with chemical cross-linking. This
yields a predominant interaction between kinesin and
β tubulin [46•,54], but interactions are also observed
with kinesin and the carboxy-terminal regions of both α
and β tubulin, possibly because of the high flexibility of
these regions [78].
The stability of the neck (and the possibility of its melting during motility) has been studied using mutants that
either strengthen or weaken the coiled-coil interaction.
As mentioned above, only the second half of the neck
helix of kinesin (residues ~355–370) has a leucine zipper
sequence providing strong interaction, whereas the first
half contains nonstandard residues not conducive to
coiled-coil formation [47]. This is consistent with results
on model peptides studied by circular dichroism
[44•,45], and with the interaction between kinesins with
varying neck lengths which dimerize only when the
necks become sufficiently long [43]. To test the interaction, the neck was altered by inserting an additional
leucine zipper sequence in order to prevent melting during
motility,
or
conversely,
by
inducing
non-helix-forming residues to weaken the interaction
([49•]; Hackney D, personal communication). The influence on motility was not as predicted, leading to the
proposal that the neck helix could remain intact and
instead the linker region on the advancing head could
flip over during movement (see Figure 3aii). A certain
length of neck must be present and flexible in order to
generate movement [79]. The caveat in the interpretations of these results is that the kinesin–microtubule
interaction may override that between the kinesin necks.
For example, there is a conformational change in the
vicinity of the motor–neck junction of Ncd upon microtubule binding, corresponding to –75 kJ/mol, which
could override the energy of coiled-coil interactions [80].
In related experiments it was shown that the region
between the neck helix and the following stalk helix
(residues ~380–405) is important for motility, rather than
merely acting as a flexible hinge [81•].
A particularly intriguing problem is that of
directionality —why can kinesin-like motors move either
Structures of kinesin and kinesin–microtubule interactions Mandelkow and Hoenger
in the plus-end or in the minus-end direction? Earlier
experiments had suggested that this property did not
depend on the position of the motor domain in the chain
(amino-terminal, middle, or carboxy-terminal), and that it
was an intrinsic property of the motor domain [82].
Recently, however, a refined analysis of the sequences
just adjacent to the core motor domain (the linker)
showed that they contain key residues that are important
for the direction. The core motor domain of a plus-motor,
coupled to the linker of a minus-motor, would move in
the minus direction, and vice versa [32••, 40••,41•,42••].
It remains to be seen whether these ‘gearbox’ residues
are responsible for the differences in dimer arrangement
in solution or on the microtubule (Figures 1a and 3a), and
how they reverse the direction of movement.
The above list is not exhaustive, there are many other constraints and interactions necessary to consider in order to
understand kinesin’s motility. We only mention the discovery of the first kinesin-specific inhibitor [83•], and the
function of the kinesin tail and the kinesin light chains
which not only connect to the cargo [3], but can also influence the motor properties [84,85].
Implications for models of motility
What do the structures tell us about models of motility?
Basically, they provide boundary conditions and a framework for discussing the mechanism of motility, but do not
explain it. We know only two reference states, the crystalline state and the microtubule-bound state. For kinesin,
the crystalline state of the dimer is probably close to the
state in solution since solution X-ray patterns are compatible
with the crystal structure [48]. The microtubule-bound state
is analogous to ‘rigor’ in actomyosin: the microtubule is completely saturated with motors in a nonmotile state, that is in
the absence of nucleotide or with nonhydrolyzable ATP
analogues. Such a periodic arrangement is required for
image reconstruction methods but is not representative of
the situation in cells where even single kinesin molecules
can generate movement. The dimeric structure of kinesin,
considered together with its processivity, has led to the dominance of hand-over-hand models of stepping, analogous to
human walking. A caveat to this interpretation is the diversity of kinesins, including even monomeric kinesins moving
at similar speeds [86•]. It is therefore possible that there are
different mechanisms of movement (such as ‘limping’, ‘hopping’, etc.). The concept of walking is reinforced by the
observation of discrete steps of 8 nm, consistent with the
‘stepping stones’ of tubulin heterodimers. If we accept the
hand-over-hand model for the sake of discussion, several
conclusions can be drawn:
There must be a point in the cycle where both kinesin
heads are attached to the microtubule at successive binding sites. At that moment the structure could be similar to
that of Figure 3bi (ring connecting the two carboxy-terminal helices). Since both kinesin and Ncd have tightly
packed heads in solution, it follows that both dimers must
41
be able to split apart and change orientation in order to
bind to microtubules in an equivalent fashion, 8 nm apart.
It is possible that Figure 3bii represents a state just before
the separation of heads would occur, but in any event the
solution structures are very different from the microtubule-bound structures.
The molecular motor paradigm is myosin which is thought
to exert force by a lever arm movement [53,87]. This has
recently been demonstrated structurally [88]. By contrast,
the corresponding neck helix of kinesin is not likely to act
as a lever because it appears only loosely bound to the core
motor domain (via the linker), and because it does not have
the proper orientation — tangential rather than perpendicular — on the microtubule surface (Figure 3aii). Thus,
force transmission by kinesin would be expected to be different from myosin.
The 8 nm step size of kinesin refers to the center of mass
movement of the whole complex. If both motors of the
dimer are involved in walking, each individual step of a
head must be 16 nm in order to generate an 8 nm centerof-mass displacement. The two heads must therefore be
able to splay apart even further than in the model of
Figure 3bi, possibly including the hinge region following
the neck. Note that each of these 16 nm steps would be
accompanied by the consumption of one ATP molecule.
Another consequence, not readily visible in Figure 5a, is
that during walking the motor heads must overtake each
other on different sides, as in human walking, because otherwise the neck and stalk would become entangled. The
two identical heads have to do two different stepping
motions to achieve a smooth processing.
Finally, we note that the discussions of motility models are
entirely centered on motors whereas microtubules are
merely regarded as passive tracks or stepping stones.
Earlier work on motor decorated sheets, however, suggested induced conformational changes in the motor-bound
tubulin subunit [22,24•]. Figure 3bi suggests that there is
an intimate contact between kinesin and tubulin, compared with the wide separation of the kinesin heads. It is
therefore conceivable that some of the information and
force transfer occurs through the microtubule. Since the
structure of tubulin is known [36••], it should become possible to design experiments to test this hypothesis.
Alternatively, since tubulin is not readily accessible to
mutagenesis, it might be possible to exploit the homologies in structure and possibly function with bacterial
proteins, such as FtsZ (homologous to tubulin [89]) and
mukB (homologous to kinesin [90]).
Acknowledgements
We thank M Thormahlen, YH Song and E-M Mandelkow for discussions,
and E Sablin and R Fletterick for providing the coordinates of dimeric Ncd
prior to publication.
42
Cytoskeleton
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