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Structure and dynamics of molecular motors
239
Structure and dynamics of molecular motors Linda A Amos* and Robert A Crosst The structures of the oppositely directed microtubule motors kinesin and ncd have been solved to atomic resolution. The two structures are very similar and are also homologous to myosin. Myosins and kinesins differ kinetically but, tantalizingly, cryoelectron microscopy has recently revealed that both structures may tilt during ADP release. Such evidence suggests that the two motor families use common structural mechanisms.
Addresses *MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK; e-mail: [email protected] '~Marie Curie Molecular Motors Group, The Chart, Oxted, Surrey RH80TE, UK; e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:239-246 Electronic identifier: 0959-440X-007-00239 © Current Biology Ltd ISSN 0959-440X Abbreviations 2D two-dimensional 3D three-dimensional AMP-PNP 5"-adenytytimidodiphosphate Pi phosphate Vi vanadate
Introduction Kinesins and myosins arc molecular motors that hydrolyse ATP in order to translocate on microtubules or actin filaments, respectively, while transporting a wide range of cellular components. Atomic structures have recently been solved for the motor domains of two molecules belonging to the kinesin family, kinesin itself [1"'] and ncd (the product of the Drosophila nonclaret disjunctional gene) [2"]. These two motors move in opposite directions on microtubule tracks, yet they have highly similar structures. The structural homology is not particularly surprising in view of their 40% sequence identity. What is startling is that these - 4 5 kDa proteins are also structurally related to the much larger motor domains of myosin, which means that our understanding of both families of motor molecules can be enhanced by comparing and contrasting them [3-6]. With the information gathered recently by means of
X-ray crystallography, 3D electron microscopy and kinetic studies, it is now possible to begin thinking about detailed models for how motor molecules move.
Motor mechanism is a structural problem Myosin and kinesin move because local conformational changes at their ATPase active sites are linked to other parts of the motor domains [3-6,7"',8]. One set of linkages must connect to the tubulin- or actin-binding regions, affecting the binding affinity of the motor domain. Nucleotide turnover causes the motor to cycle between weak binding conformations, which detach from the track, and strong binding conformations, which hold force (Fig. la, Table 1). The linkages between the ATPase and track-binding sites work reciprocally, in that the binding of tubulin or actin produces a signal in the reverse direction, altering the affinity of the active site for nucleotides and, in particular, speeding up the process of product release (Table 2). A second set of linkages connects to the load, allowing conformational changes in the track-motor complex to exert useful force. Again, the linkages work reciprocally, so that pulling on the motor molecule affects the affinity for both the nucleotides and the track (the so-called 'Fenn effect') [9,10]. The problem is understanding the connectivity: how the active site, the track-binding site and the load-binding site communicate.
Myosin and microtubule motors have homologous structures Myosin motor domains have nearly double the mass of the kinesin motor domain and the two families show little sequence homology. Nevertheless, they contain essentially the same basic core structure, consisting of a 7-8 stranded 13 sheet, sandwiched between two sets of three oc helices [1"]. Only one 13 strand within the cores of kinesin and ned cannot be aligned with a similar feature in myosin. The major differences occur in stretches of polypeptide that loop out from the cores, the greater size of myosin being due to much larger loops (Figs lb,2). Even within these loops, the elements involved in binding to microtubules or actin filaments appear to be homologous (see below) [1",2"].
Table 1 Chemical coupling constants for kinesins and myosin*. Motor
Motor alone (~M)
Motor.ATP (I.tM)
Motor.AMP.PNP (I.tM)
Motor.ADP.Pi (jiM)
Motor.ADP (/aM)
References
Kinesin ncd Eg5 Myosin
0.002 0.012 1 0.01-0.1
? ? ? 100
0.13 0.12 0.02 105
? 0.5f ? 50
6 6 > 10 10
[49] [49,50] [51 ] [10]
*Track dissociation constants (per actin monomer and tubulin heterodimer) are given for kinesin, ncd (a reverse-directed homologue), Eg5 (a slower homotogue) and myosin. ;BeF complex. SVery dependent on ionic strength.
Macromolecular assemblages
240
Figure 1
(a)
(b)
Weak binding nucleotide trapped
Strong binding nucteotideex~anges
Myosin S1 50kD I 20kD 1olo I 3ol0 4o01 I I 1 I I 200 500 eoo too 800 Proximal domain of motor - Distal domain of motor ~ Lever arm 25kD
0I
[
I
m:i •
•
i;
Insedion 1 A1
~'~
KoADP M-ATP M.ADP.Pi
K. . . . . K * A M ~ P N P [=ATP?] K.AD~Pi M. M.ADP
Kinesin motor d o m a i n l 1 N4 ~-,~ 295 335
m~:m~
"~ l l "
A2
IIL~ L III1~1
N1
N2
l ~ l ~i ~,2
• ::: *t3
N3 L12 I I I c~4 (~5 c~6
Insertion 2 A
l;
I ELC RLC
~,~l 340
401
I
Ncd motor domain
700
Functional and structural relationships between myosin and kinesin motors. (a) Cyclic transition of binding states between motor and track. Motor domains (K, kinesin; M, myosin) cycle between weakly bound and strongJy bound conformations, depending on whether nucleotide (solid black circle) is present or not in the active site. Both families of motor are strongly bound when the site is empty, but the transition steps between weak and strong binding differ (see Table 1). A kinesin domain remains strongly attached to tubulin whilst cleaving Pi from ATP and releasing it; following detachment kinesin becomes strongly bound again by releasing ADP. Myosin is immediately detached from actin on binding ATP and reattaches strongly by releasing Pi. Kinesin only releases ADP readily when interacting with microtubules; myosin only releases Pi readily when interacting with actin filaments (see Table 2). (b) Homology in the arrangement of sequence segments. Schematic comparison of the amino acid sequences of the $1 fragment of myosin and the motor domains of kinesin and ncd. c~-helical segments are shown in black, ~-sheet strands are shown in grey and the putative coiled-coil extensions from kinesin and ncd motor domains are shown as cross-hatched segments; the latter segments were absent from the proteins that were crystallized [1"°,2"] but were present in dimeric motors studied by electron microscopy [29"',32"]. The scale at the top shows amino acids in the myosin sequence numbered from the N terminus. The 25, 50 and 20 kDa fragments obtained by proteolysis are also marked. A1 (405-415), A2 (529-558) and A3 (626-64?) labe{ points in the myosin sequence that are believed to contact actin [16]. ELC and RLC refer to the essential and regulatory light chains of myosin II, which are associated with the long ~-helical lever arm or 'regulatory domain' (?62-842). Insertion 1 in the myosin sequence seems to be functionally equivalent to loop L8 of kinesin or ncd; insertion 2 seems to be equivalent to loop L12. Small loops at N1-N4 are responsible for binding nucleotide to kinesin or ncd; N2 and N3 are also known as 'Switch 1' and 'Switch 2' [?°°]. AMP.PNP is a nonhydrolysable analogue of ATP. Figure 1 b adapted with permission from [1"'].
Table 2 Kinetic constants for kinesins and myosin'. Motor Kinesin -MTs Kinesin + MTs ncd - Mrs ncd + MTs Eg5 - MTs Eg5 +MTs Myosin -F-actin Myosin + F-actin
Hydrolysis (s-1)
Pi release (s-1)
ADP release (s-1)
Turnover (s -1)
References
9 1O0
>10 >13 ~ ? ? ? ? 0.1 50-1 O0
0.002-0.01 295, 300 0.003 1.65 0.006 4.94 1 300-500
0.02 20, 275 0.002 1.1, 0.885 0.01 1.42, 1.925 0.05 10-1 O0
[52-55] [52-54,56] [42,56,5?] [42,56,57] [5 t] [51] [10] [10]
50-1 O0 50-100
*Rates for catalysis (two-headed motors, rates per site) are given for kinesin, ncd (a reverse-directed homologue), Eg5 (a slower homologue) and myosin. MTs, microtubules. )Equal to cycle rate. STurnover of mant-ATP (a fluorescent analogue of ATP).
Motions in homologous active sites actuate different events Mg.ATP hydrolysis within the proteins involves the cleavage of the y-phosphate bond followed by the release of phosphate (Pi) and subsequently Mg-ADP (Fig. la). The effects of the various nucleotide intermediates on track binding, and the effects of track binding on ATP turnover, differ substantially amongst the three motors (Tables 1,2). Specifically, the transitions from strong to
weak track binding occur at different points in the cycle. Myosin is unusual in that Mg-ADP.Pi is trapped in the active site in the absence of actin [11]. Actin binding releases Pi (possibly through a 'back door' [12]) and the motor locks down on to the track. Pi is rapidly released from kinesin and ned, but Mg.ADP is trapped in the absence of mierotubules [8,9]. Microtubule binding triggers Mg.ADP release and locks the motor down. In both cases, tight binding to the track is coupled to an
Structure and dynamics of molecular motors Amos and Cross
active-site conformational change that releases the trapped products and allows Mg.ATP rebinding. This ensures that nucleotide turnover is tightly coupled to track binding.
Conformational changes associated with nucleotide turnover in myosin There are good grounds for believing that Pi release triggers the major force generating conformational change in myosin [10] but attempts by electron microscopy to visualize a structural change have been repeatedly frustrated, because the weak binding myosin.Mg.ADP-Pi
941
starting state is both difficult to populate and disordered; the myosin molecules that do bind to actin seem to be
attached at many different angles. However, recent 3D image reconstruction from electron micrographs of some types of myosin S1 heads bound to F-actin [13",14 °] has revealed some domain motion coupled to Mg.ADP release. A 'lever arm' (see Fig. 2) of the myosin motor moves substantially, on the release of Mg.ADE in both brush border and smooth muscle myosin; however, no such movement appears to occur in rabbit skeletal myosin when Mg-ADP is released [15].
Figure 2 (a)
Distal domain of N motor
Lever arm
Actin contact in Insertion 2 Actin contact in Insertion 1
(b)
p,
c;, 1997 Current Opinion in Structural Biology
Comparison of crystal structures. Corresponding views of (a) myosin and (b) kinesin, looking down on the nucleotide (shown in solid black; see also Fig. 5 of [7"]). The structure of ncd (not shown) is almost identical to that of kinesin [1 °°,2°*]. The homology in the structural elements that support the nucleotide-binding pocket is less apparent than in views at right angles to this [1°°], but the myosin domains that move relative to one another [11,16] are more distinct. The distal domain (residues 45'77-762) appears to rotate around a hinge near residue 45'7 (marked by asterisk). SH1 and SH2 indicate two cysteines that can be cross-linked in solution [21,22]; their large separation in all the available crystal structures of myosin suggests that only part of the full conformational change has been shown. Insertion 1, consisting of loop L8 with strands ~5a and ~5b, and Insertion 2, consisting of loop L12, are predicted to make contact with tubulin (see Fig. 3c) [1 °°]. The crystal structure of kinesin lacks a short segment (residues 325-332) at its C terminus (shown by arrow head); the structures of this segment and the subsequent stretch of o~ helix are therefore hypothetical ['7°°].
249
Macromolecular assemblages
T h e initial evidence for relative movement of myosin domains came from fitting atomic structures into a 3D map derived by image reconstruction from electron micrographs of frozen-hydrated actin filaments complexed with myosin S1 [16]. In order to fit the crystal structures of myosin that contains bound nucleotide into the rigor structure (containing no nucleotide) imaged in the complex, it was necessary to close a cleft between two segments, referred to in Figures lb and 2 as Insertion 1 and Insertion 2. Rayment et al. [16] suggested that the initial weak binding of myosin.Mg-ADP.Pi to actin might be via a site in Insertion 2, and that this interaction might be followed by closure of the cleft and formation of an additional interaction between actin and a site in Insertion 1 (see also [17,18]). Crystal structures of the Dictyoste/ium myosin motor domain [11,19°,20 °] provide more direct evidence for nucleotide-dependent conformational changes in myosin. Two conformations have been observed: the BeF 4 and pyrophosphate complexes are thought to be analogues of a myosin.ATP transition state, whereas the AIF 4 and ADP-Vi complexes are more myosin.ADP.Pi-like. T h e transition from the ATP-like to ADP-Pi-like states partially closes the cleft between Insertion 1 and Insertion 2 (Fig. 2). This transition elicits a response in a so-called 'converter region' in the neck of the molecule. T h e crystallographic evidence is very clear, but the motions in solution may be more dramatic: two cysteines known as SH1 (Cys697) and SH2 (Cys707), which can be cross-linked in the ADP.Vi state in solution [21,22] (thereby trapping ADP.Vi in the active site), are still 18 ~ apart in the crystal structures, at opposite ends of a kinked c~ helix between the active site and the lever arm (Fig. 2a).
Lever arms as amplifiers T h e motion of a lever arm is clearly an effective way to amplify more modest motions within the myosin head [23]. Mutagenesis experiments in which extra copies of the light chain binding regions were concatenated to the natural sequence have established that the sliding velocity at pseudozero load correlates with increases in lever arm length [24]. It is not clear, however, how stiff the lever arm is. T h e replacement of the authentic lever arm with ct-actinin, which is predicted to be stiff, preserves the unloaded sliding velocity [23]. Howard and Spudich (in an appendix to [24]) considered the possibility that the lever is flexible. To what extent the lever arm functions as a genuine cantilever or as a flexible tether is unclear. Myosin light chain mediated regulation may possibly work by switching the stiffness of the lever arm. Motility assays carried out under load will be necessary to investigate these points.
Conformational changes in kinesin and ncd T h e recently solved atomic structures for kinesin and ncd [1°°,2°°] are the motor.ADP states, which are very stable
[Table 1]. No crystallographic information is available about other states, and the clearest clues to how the active sites of kinesins may move derive from work on the Ras GTPase (for reviews, see [2°°,7°°]). T h e GTPase site of Ras is homologous to the myosin and kinesin ATPases sites. For Ras and other homologous G-proteins, two small loops, named Switch 1 and Switch 2, face each other across a cleft into which the y-phosphate of the trinucleotide would protrude when present. T h e conversion from the G T P to the G D P complex induces a progressive scissor-like motion between Switch 1 and Switch 2. Vale [7 °°] has suggested that dinucleotide is subsequently released because of a subtle change in the Switch 2 region that causes the Mg2+ ion to be lost. Dinucleotide release switches kinesin into a strong binding (force-holding) conformation and presumably does so by producing some further movement around the nucleotide-binding pocket. Exactly what happcns is not clear. In contrast to the myosin situation, the connectivity between the nucleotide-binding site of kinesin or ncd and the microtubule-binding sites is only just beginning to be elucidated using mutagenesis. An ncd point mutation, on one of the [~ strands between the proposed tubulin-binding surface and the nucleotide, slows ATP turnover and affects microtubule affinity [25]. T h e 3D electron microscopy of monomeric kinesin motor domains bound to microtubules (see Fig. 3a) [26"] indicates that A D P release is coupled to an angular change in one portion of the motor domain. T h e underlying tubulin structure appears to be essentially unchanged [27], which is consistent with the observation that kinesincoated beads are still able to move on microtubules that have been cross-linked with glutaraldehyde [28]. How this relatively modest candidate power stroke might be amplified to produce the 16nm strides necessary for processivity [9,29"] is discussed below.
Interaction between kinesin and microtubules T h e definitive fitting of the atomic structures of kinesin and ncd into 3D electron microscope images of these motor domains bound to the surfaces of microtubules or tubulin sheets [29"°,30,31,32 °] will probably require images of frozen-hydrated specimens labelled with electron-dense markers. Information from crystal structures of dimeric motors, which are likely to be solved in the near future, should greatly restrict the range of possible orientations. Meanwhile, it is possible to make some tentative guesses. Figure 3b shows the kinesin crystal structure oriented so that the two putative tubulin-binding loops L8 and L12 [1*°,2*',7 °°] lie on the reverse side and, in Figure 3c, this view has been fitted to electron microscope images in two possible orientations. In both cases, the two loops are positioned on the face of kinesin that would contact tubulin and are roughly one above the other; however, the prominent spike observed on the left side of heads
Structure and dynamics of molecular motors Amos and Cross
Figure 3
243
[33-35,36"] relative to those in normal microtubules [36"',37,38]. According to consensus, features identifying the N and C termini of the tubulin monomers [33] lie in the left half of a protofilament, as viewed from outside a microtubule oriented with its plus end upwards, and close to where kinesin and ncd motor domains appear to bind (see Fig. 3a,c). Independent evidence suggests that kinesin binds near to the C termini of tubulin monomers [39,40].
The kinesin directionality problem
Fitting the kinesin crystal structure to 3D electron microscope images. (a) Part of a model combi~ning views of a microtubule decorated with monomeric kinesin motor domains in three different nucleotide states (AMP.PNP, no nucleotide [nucl], ADP; see also Fig. 3e of [27]). The individual 3D models are calculated from electron microscope images of negatively stained specimens [26°]. A prominent spike, on the left of each motor domain in this view, appears to rotate towards the microtubule plus end (oriented towards the top of the page) when Mg.ADP is lost from its nucleotide-binding site. Binding of AMP-PNP, a nonhydrolyzable analogue of ATP, does not cause a reverse movement. The vertical spacing of the motor domains corresponds to the 8 nm length of tubulin heterodimers. (b) A view of the kinesin motor domain at about 90" to that in Figure 2b, rotated so that Insertions 1 and 2 (see Figs lb,2b) are on the reverse side. In this orientation, both the N and C termini of the traceable regions of the polypeptide are towards the top of the page; there are six unseen residues at the N terminus and 24 at the C terminus. Figure redrawn from a view provided by J Kull. (c) A model obtained by combining several 3D electron microscope images of frozen-hydrated microtubules decorated with motor domains [29°°]. The longitudinal 'protofilaments' of the microtubule show a 4 nm periodicity corresponding to the tubulin monomer spacing. Attached to some sites on the protofilaments are images of monomeric kinesin (K), dimeric kinesin (attached domain, K1 ; tethered domain, K2) and dimeric ncd (N1 and N2). Superimposed on images of two kinesin monomers are miniaturised views of the kinesin crystal structure (one oriented as in Fig. 3b, the other rotated 135"). The side views on the left (based on Fig. 2b) show loops L8 (incorporating 1~5aand I]5b) and L12 binding separately to tubulin; the hypothetical dimer structure illustrates the proposal that the two strands of the coiled coil may separate to allow the tethered motor domain to find the next available binding site [8,9].
in the electron microscope images fits into two alternative features in the atomic structure. Recent reports agree about the probable orientation of tubulin protofilaments in Zn-induced 2D crystals
In 3D images of kinesin and ncd motor domains bound to tubulin [25,26",27,29,30], the two species of attached motor domains are almost indistinguishable but the second, unattached, heads of the dimeric molecules are differently oriented (Fig. 3c). The second head of a kinesin dimer, joined to the attached head at its C terminus, is oriented towards the plus end, whereas the second head of ncd, joined to its partner at the N terminus, points towards the minus end. The appropriate tilting of the free head by the bound head, as observed by electron microscopy, will clearly help to specify directionality by directionally biasing the free head. This is not in itself enough, however, because kinesin single heads are directional also. Moreover, the direction in which kinesin motors push microtubules has been shown to be unchanged by moving the tail from the C to the N terminus [41]; therefore, differences within the motor probably determine the directions of motility. What sort of differences? The main structural differences occur within a group of three small loops, L1, L5 and L9, that lie on the points of a triangle enclosing the nucleotide, and within two slightly larger loops, L2 and L l l , on either side of the y-phosphate cleft (see Figs 2b,3b). Vale and coworkers [2"°,7 °'] have suggested that the loss of y-phosphate from kinesin or ncd triggers the opposite movement of a structural unit composed of L l l and c~ helix 4 in each protein. Alternatively, Lockhart and Cross [42] have suggested that the key to directionality lies in the angle of attack of the weak binding state, in which each motor domain first attaches to tubulin (illustrated in Fig. 4). On the basis of its structural homology with myosin, we expect kinesin and ned motor domains to make their initial attachments to tubulin using just one of the two putative tubulin-binding loops. If nucleotidc hydrolysis and product release are sensed by the core structures and transmitted to a pair of tubulin-binding sites on the surface, small sequence changes within the binding sites might favour initial binding by one site rather than the other and thereby reverse the direction. In our view, this suggestion remains the most plausible candidate mechanism by which two such similar structures can develop force in opposite directions. Thus, we suspect that the initial recognition of microtubnles by the motor-Mg-ADP states of kinesin and ncd operates via different surface loops, possibly L8
244 Macromolecul assembl ar ages for kinesin and L12 for ncd. This idea should be testable using mutagenesis.
Figure4 (a)I
~
(b)
QI
site, which, quite clearly, requires some unzipping of the coiled coil via which the two motor domains associate (see Figs 3c,4a) [8]. The tethered head would then be free to diffuse randomly in search of a new binding site within the volume allowed by its tether; it will obviously be helpful if the search pattern is biased by a suitable tilting of the site to which the tether is attached. Reconciling the fixed positions of kinesin head 2 observed by electron microscopy with this prediction of mobility is a problem, but Hirose et al. [29"'] have suggested that head 2 exchanges rapidly between parked and freely diffusing states. Several groups are actively mutating the neck region of kinesin to seek an effect on processivity. There is evidence of a similar unzipping in myosin [44,45], which may allow each motor domain to search independently for a new binding site.
Synthesis: power strokes and Brownian ratchets
1997 Current Opinion in Structural Biology
Motility schemes for kinesin and ncd. (a) The processive behaviour of kinesin may possibly be explained by melting of part of the coiled-coil structure through which the motor domains (heads) are paired [8,9]. Thermal motion will then allow the tethered head to search for the nearest available binding site (stage I). One of the tubulin-binding loops binds first (stage II). After release of Mg.ADP and attachment of the second loop, the leading head is strongly attached (stage Ill). Because of the conformational change associated with ADP loss, the lead head exerts a tension on the rear head; the effect of tension is highly directional, causing the rear head (but not the leading head) to detach (stage IV) and it is pulled forwards to begin the next cycle. (b) In order to explain the different directions of kinesin and ncd movement, we suggest that initial attachment is via different loops (stage II), before settling down into the strongly attached state (stage III). In addition, on the one hand, when a kinesin head is fully attached, it needs to be pulled off much more easily by its partner pulling from the plus direction than by tension from the minus direction. On the other hand, ncd needs to resist tension from the plus direction when pulling a load towards the minus end.
The processivity mechanism of kinesin Vale et al. [43] have shown that untrapped fluoresecently labelled single (dimeric) molecules of kinesin are processive, neatly removing lingering doubts that earlier observations of processivity by optical trapping were due to the trapping scheme. Kinesin tracks microtubule protofilaments efficiently [9] and in all probability, moves 'hand over hand' in 8nm steps along a single protofilament. In order to do this, each motor domain needs to detach and swing forward 16nm to its next binding
Processive stepping by kinesin must involve a substantial diffusional component, and this concept is also influencing thoughts about the myosin mechanism [23]. We suggest that the generalized motor depicted in Figure 4a carries out a diffusional scan for its binding site using a surface loop as a lasso and subsequently locks down using a conformational change (a power stroke). The progress (step distance) is equal to the amplitude of the conformational change plus the distance diffused. The balance between the different contributions (diffusion versus power stroke) may be different between myosins and kinesins, but a role for biased diffusion in the myosin mechanism seems reasonable. The longitudinal step from one actin monomer to the next is 5.5 nm, but measurements using optical trapping experiments suggest that the shift produced may be as little as 2nm [46]. Perhaps the implausibly long lever arms in some vesicular myosins [47] imply processivity?
Dynein The study of the structure and function of dynein, another type of microtubule-associated motor, has scarcely begun compared with the progress made for the myosin and kinesin families. Kinetically, dynein appears to behave in a similar way to myosin [8]; however, a dynein molecule usually has a pair of motor domains, each at least five times the size of a kinesin motor domain, and the structural changes involved in motility are probably complex. A recent study has yielded evidence suggesting that active molecules shift by as much as 12nm during a kinetic cycle [48"].
Conclusions The elucidation of motor mechanism is one of the most challenging problems in biology, yet enormous progress has been made in the past year. Apparently, the movements made in the active sites of myosin, kinesin and ned are fundamentally homologous but each species of motor amplifies them differently. We have proposed,
Structure and dynamics of molecular motors Amos and Cross
in particular, that the two distinct tubulin-binding regions of kinesin and ncd may bind sequentially; and that the direction of morion may depend on which of the two regions binds first. We expect rapid progress in the field as this and other possible model amplification mechanisms are tested.
245
muscle myosin) about the end of the actin-binding domain, which remained unchanged. 14. •
Whittaker M, Wilson-Kubalek EM, Smith JE, Faust L, Milligan RA: A 35A movement of smooth muscle myosin on ADP release. Nature 1995, 378:748-751. See annotation [13"]. 15.
Gollub J, Creme CR, Cooke R: ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin. Nat Struct Bio/1996, 3:796-802.
Acknowledgements
16.
Molecular motors arc a large subject and the cxit~encies uf space have unfortunately prevented us citing everything and c v c u o n e we wished to. ",~e arc, however, especially gratcfut to Jon KulI f'~r pro~ iding images of the crystal structures; other ~iews of kincsin and ned can be seen at his web site [58].
Rayment I, Holden HM, Whittaker M, Yohn CB, Lorenz M, Holmes KC, Milligan RA: Structure of the actin-myosin complex and its implications for muscle contraction. Science 1993, 261:58-65.
1 "7.
Uyeda TO, Ruppel KM, Spudich JA: Enzymatic activities correlate with chimaeric substitutions at the actin-binding face of myosin. Nature 1994, 368:567-569.
18.
Ruppel KM, Spudich JA: Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft. Mol Biol Ce//1996, 7:1123-1136.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •,
of speciat interest of outstanding interest
1. •.
Kull FJ, Sablin EP, Lau R, Fletterick RJ, Vale RD: Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature 1996, 380:550-555. The atomic structure of the human kinesin motor domain is revealed to be an arrowhead-shaped domain with a few loops on its surface. The cluster of (x helices and ]3 strands forming the main domain turns out to be much more similar to the core of the myosin motor domain than was expected. 2. ..
Sablin EP, Kull FJ, Cooke R, Vale RD, Fletterick RJ: Crystalstructure of the motor domain of the kinesin-related motor ncd. Nature 1996, 380:555-559. In this paper, the crystal structure of the Drosophila motor ncd with bound Mg.ADP is compared with the kinesin structure [1°*], showing that the two motor domains are remarkably similar despite their opposite directions of movement. A model is proposed in which kinesin and ned react in opposite ways to the presence or absence of the cleavable y-phosphate of ATP.
19. •
Fisher AJ, Smith CA, Thoden JB, Smith R, Sutoh K, Holden HM, Rayment I: X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with Mg.ADP.BeF(x) and Mg.ADP.AIF4-. Biochemistry 1995, 34:8960-8972. See annotation [20"]. Smith CA, Rayment I: X-ray structure of the magnesium(ll).ADP vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. Biochemistry 1996, 35:5404-5417. These two papers [19",20"] report the crystal structures of a truncated myosin head complexed with Mg.ADP and possible phosphate analogues. The absence of most of the (x-helical neck or lever arm and its associated light chains allowed the globular motor domain to be crystallized without the modifications required for crystallization of complete $1 fragments. Structural changes observed between different nucleotide states are thus more likely to be functional. The observed response to a change in bound nucleotide is a significant rotation of the distal domain (residues 457-?62) relative to the domain that binds to actin. 20. •
21.
Burke M, Reisler E: Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin: chemical probing of movement of residues during conformational transitions. Biochemistry 1977, 16:5559-5563.
22.
Wells JA, Yount RG: Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1. Proc Nat/Acad Sci USA 1979, 75:4966-4970.
3.
Sakowicz R, Goldstein LSB: The muscle in kinesin. Nat Struct Biol 1996, 3:404-407
4.
Sellers JR: Kinesin and ned, two structural cousins of myosin. J Muse Res Cell Moti11996, 17:1 73-175.
5.
Hackney DD: Myosin and kinesin: mother and child reunited. Chem Bio11996, 3:525-528.
23.
Block SM: Fifty ways to love your lever: myosin motors. Ceil 1996, 87:151-157.
6.
Rayment I: Kinesin and myosin: molecular motors with similar engines. Structure 1996, 4:501-504.
24.
Uyeda TOP, Abramson PD, Spudich JA: The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc Nat/Acad Sci USA 1996, 93:4459-4464.
25.
Moore JD, Song H, Endow SA: A point mutation in the microtubule binding region of the ncd motor protein reduces motor velocity. EMBO J 1998, 15:3306-3314.
Vale RD: Switches, latches, and amplifiers: common themes of G proteins and molecular motors. J Ceil Biol 1996, 135:291-302. This stimulating review compares and contrasts the structures and activities of myosin, kinesin and ned with severa/G proteins whose atomic structures are known. All have a recognizably similar fold around the nucleotide, especially near the y-phosphate. Pre-existing information about the reaction of G proteins to trinucleotide hydrolysis sheds light on the probable mechanisms of motor proteins. 7. o•
Hirose K, Lockhart A, Cross RA, Amos I_A: Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. Nature 1995, 376:277-279. 3D electron microscope images of negatively stained specimens show 16protofilament microtubules from cricket sperm, decorated with monomeric kinesin motor domain with variations in bound nucleotide. A small projection from the motor domain appears to be tilted nearer to the microtubule plus end in the nucleotide-free state, as compared with the Mg.ADP-containing state. 26. •
8.
Hackney DD: The kinetic cycles of myosin, kinesin, and dynein. Annu Rev Physiol 1996, 58:731-750.
9.
Howard J: The movement of kinesin along microtubules. Annu Rev Physiol 1996, 58:703-729.
tO.
Bagshaw CR: Muscle Contraction, edn 2. Chapman Hilh New York; 1993.
27.
Amos LA, Hirose K: Microtubule-motor complexes. Curt Open Ce// Bio/1997, 9:4-11.
11.
Rayment I, Smith C: The active site of myosin. Annu Rev Physiol 1996, 58:671-702.
28.
Turner D, Chang C, Fang K, Cuomo P, Murphy D: Kinesin movement on glutaraldehyde-fixed microtubules. Anal Biochem 1996,242:20-25.
12.
Yount RG, Lawson D, Rayment I: Is myosin a 'back door' enzyme.'? Biophys J Suppl 1995, 68:44-49.
Jontes JD, Wilson-Kubalek EM, Milligan RA: A 32" tail swing in brush border myosin I on ADP release. Nature 1995, 378:751-753. These two papers, 113*,14"] compare 3D maps of F-actin decorated with myosin heads in the presence and absence of Mg-ADP. The C-terminal light chain binding domain was reported to pivot 32" (or 23" in the case of smooth 13. •
Hirose K, Lockhart A, Cross RA, Amos LA: Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules. Prec Nat/Acad Sci USA 1996, 93:9539-9544. This paper compares the two oppositely moving double-headed motors in the AMP-PNP state. The attached head of each dimer resembles singleheaded kinesin and binds to tubulin in the same way; the positions of the second heads are quite different. These are the first reported structural dif29. •,
246
Macromolecular assemblages
ferences that appear to be relevant to the opposite directions of movement of the two motors. In addition, the connections between pairs of heads allow the approximate positions of both the N and C termini to be identified in an attached head.
41.
Stewart RJ, Thaler JP, Goldstein LS: Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein. Proc Nat/Acad Sci USA 1993, 90:5209-5213.
30.
Sosa H, Milligan RA: Three dimensional structure of ncd decorated microtubules obtained by a back-projection method. J Mo/Bio/1996, 260:743-755.
42.
Lockhart A, Cross RA: Origins of reversed directionality in the ncd molecular motor. EMBO E 1994, 13:751-757.
31.
Hoenger A, Milligan RA: Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. J Mo/ Bio/1997, 265:553-564.
43.
Vale RD, Fumatsu T, Pierce DW, Romberg L, Ilarada Y, Yanagida T Direct observation of single kinesin molecules moving along microtubules. Nature 1996, 380:451-453.
44.
Offer G, Knight P: The structure of the head-tail junction of the myosin molecule. E Mo/Bio/1996, 256:407-416.
45.
Knight PJ: Dynamic behaviour of the head-tail junction of myosin. J Mo/ Bio/1996, 255:269-274.
46.
Molloy JE, Burns JE, Kenddck-Jones J, Tregear RT, White DCS: Movement and force produced by a single myosin head. Nature 1995, 378:209-212. Cope MJTV, Whisstock J, Rayment I, Kendrick-Jones J: Conservation within the myosin motor domain: implications for structure and function. Structure 1996, 4:969-987.
32. •
Arnal I, Metoz F, DeBonis S, Wade RH: Three-dimensional structure of functional motor proteins on microtubules. Curr Bio/1996, 6:1265-1270. This polarity of microtubules decorated with double-headed motors is determined in this study using microtubules assembled from centrosomes. Helically symmetrical 15-protofilament microtubules are selected from a mixed population of reassembled brain microtubules for reconstruction of 3D iraages comparing dimedc kinesin and ncd. The results agree with [29"]. 33.
Nogales E, Wolf SG, Khan IA, Luduena RF, Downing KH: Structure of tubulin at 6.5A and location of the taxol-binding site. Nature 1995, 375:424-427.
47.
34.
Ray S, Wolf SG, Howard J, Downing KH: Kinesin does not support the motility of zinc-macrotubes. Cell Motil Cytoske/ 1995, 30:146-152.
35.
Nogales E, Wolf SG, Zhan SX, Luduena RF, Downing KH: Preservation of 2-D crystals of tubulin for electron crystallography. J Struct Biol 1996, 115:199-208.
48. Burgess SA: Rigor and relaxed outer dynein arms in replicas of • cryofixed motile flagella. J Mol Biol 1995, 250:52-63. Electron microscope images of thousands of individual freeze-etched flagellar dynein arms are compared by single-particle computer analysis. When ATP is rapidly diluted out, the arms appear to be shifted 12nm away from their relaxed positions. In specimens frozen while active, a proportion of the arms are also in this maximally shifted position, suggesting it is a stage in the kinetic cycle. The remaining arms are either relaxed or in a slightly tilted conformation.
36. ••
Wolf SG, Nogales E, Kikkawa M, Gratzinger D, Hirokawa N, Downing KH: Interpreting a medium resolution model of tubulin: comparison of zinc-sheet and microtubule structure. J Mo/Biol 1996, 262:485-501. A difference map comparing unmodified Zn-tubulin with subtilisin-digested tubulin, lacking part of the C-terminal segment, shows a peak close to the rod-shaped feature tentatively identified in [33] as a predicted C-terminal c( helix. The plus-minus orientation of Zn-tubulin protofilaments was established by comparison of their projected density distribution with that of protofilaments in normal tubulin sheets of known polarity. An attempt to orient the 3D map into a helical reconstruction from ice-embedded microtubules did not lead to a single best fit, apparently because of a difference in protofilament thickness in the two assemblies. As the electron fibre diffraction patterns from microtubule bundles show a general similarity to the electron diffraction patterns from Zn-tubulin crystals, the most probable explanation is that the conformation of Zn-tubulin is simply more compact than normal tubulin. This difference may explain why motors do not interact with Zn-tubulin [34]. 3?.
Hoenger A, Milligan RA: Polarity of 2-D and 3-D maps of tubulin sheets and motor-decorated sheets. J Mol Biol 1996, 263:114-119.
38.
Hirose K, Amos WB, Lockhart A, Cross RA, Amos LA: Threedimensional cryo-electron microscopy of 16-protofilament microtubules: polarity and interaction with motor proteins. J Struct Bio1199"7, in press.
39.
Goldstein LSB: Structural features involved in force generation in the kinesin superfamily. Biophys J Supp11995, 68:260-266.
40.
Larcher JC, Boucher D, Lazereg S, Gros F, Denoulet P: Interaction of kinesin motor domains with alpha- and betatubulin subunits at a tau-independent binding site: regulation by polyglutamylation. J Bio/Chem 1996, 271:22117-22124.
49.
Crevei IM-T, Lockhart A, Cross RA: Weak and strong states of kinesin and ncd. J Mo/Bio/1996, 257:66-76,
50.
Rosenfeld SS, Rener B, Correia JJ, Mayo MS, Cheung HC: Equilibrium studies of kinesin-nucleotide intermediates. J Bio/ Chem 1996, 271:9473-9482.
51.
Lockhart A, Cross RA: Kinetics and motility of the Eg5 microtubule motor. Biochemistry 1996, 35:2365-2373.
52.
Gilbert SP, Johnson KA: Pre-steady-state kinetics of the microtubule-kinesin ATPase. Biochemistry 1994, 33:1951-1960.
53.
Ma YZ, Taylor EW: Mechanism of microtubule kinesin ATPase, Biochemistry 1995, 34:13242-13251.
54.
Gilbert SP, Webb MR, Brune M, Johnson KA: Pathway of processive ATP hydrolysis by kinesin. Nature 1995, 373:671-676.
55.
Hackney DD: Kinesin ATPase: rate-limiting ADP release. Proc Natl Acad Sci USA 1988.85:6314-6316.
56.
Lockhart A, Cross RA, McKillop DF: ADP release is the ratelimiting step of the MT activated ATPase of non-claret disjunctional and kinesin. FEBS Lett 1995, 368:531-535.
5"7.
ShimizuT, Sablin E, Vale RD, Fletterick R, Pechatnikova E, Taylor EW: Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain. Biochemistry 1995, 34:13259-13266.
58.
Jon Kull homepage on World Wide Web URL: http://util.ucsf.edu/people/kull/kinesin.html.
Structure and dynamics of molecular motors Linda A Amos* and Robert A Crosst The structures of the oppositely directed microtubule motors kinesin and ncd have been solved to atomic resolution. The two structures are very similar and are also homologous to myosin. Myosins and kinesins differ kinetically but, tantalizingly, cryoelectron microscopy has recently revealed that both structures may tilt during ADP release. Such evidence suggests that the two motor families use common structural mechanisms.
Addresses *MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK; e-mail: [email protected] '~Marie Curie Molecular Motors Group, The Chart, Oxted, Surrey RH80TE, UK; e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:239-246 Electronic identifier: 0959-440X-007-00239 © Current Biology Ltd ISSN 0959-440X Abbreviations 2D two-dimensional 3D three-dimensional AMP-PNP 5"-adenytytimidodiphosphate Pi phosphate Vi vanadate
Introduction Kinesins and myosins arc molecular motors that hydrolyse ATP in order to translocate on microtubules or actin filaments, respectively, while transporting a wide range of cellular components. Atomic structures have recently been solved for the motor domains of two molecules belonging to the kinesin family, kinesin itself [1"'] and ncd (the product of the Drosophila nonclaret disjunctional gene) [2"]. These two motors move in opposite directions on microtubule tracks, yet they have highly similar structures. The structural homology is not particularly surprising in view of their 40% sequence identity. What is startling is that these - 4 5 kDa proteins are also structurally related to the much larger motor domains of myosin, which means that our understanding of both families of motor molecules can be enhanced by comparing and contrasting them [3-6]. With the information gathered recently by means of
X-ray crystallography, 3D electron microscopy and kinetic studies, it is now possible to begin thinking about detailed models for how motor molecules move.
Motor mechanism is a structural problem Myosin and kinesin move because local conformational changes at their ATPase active sites are linked to other parts of the motor domains [3-6,7"',8]. One set of linkages must connect to the tubulin- or actin-binding regions, affecting the binding affinity of the motor domain. Nucleotide turnover causes the motor to cycle between weak binding conformations, which detach from the track, and strong binding conformations, which hold force (Fig. la, Table 1). The linkages between the ATPase and track-binding sites work reciprocally, in that the binding of tubulin or actin produces a signal in the reverse direction, altering the affinity of the active site for nucleotides and, in particular, speeding up the process of product release (Table 2). A second set of linkages connects to the load, allowing conformational changes in the track-motor complex to exert useful force. Again, the linkages work reciprocally, so that pulling on the motor molecule affects the affinity for both the nucleotides and the track (the so-called 'Fenn effect') [9,10]. The problem is understanding the connectivity: how the active site, the track-binding site and the load-binding site communicate.
Myosin and microtubule motors have homologous structures Myosin motor domains have nearly double the mass of the kinesin motor domain and the two families show little sequence homology. Nevertheless, they contain essentially the same basic core structure, consisting of a 7-8 stranded 13 sheet, sandwiched between two sets of three oc helices [1"]. Only one 13 strand within the cores of kinesin and ned cannot be aligned with a similar feature in myosin. The major differences occur in stretches of polypeptide that loop out from the cores, the greater size of myosin being due to much larger loops (Figs lb,2). Even within these loops, the elements involved in binding to microtubules or actin filaments appear to be homologous (see below) [1",2"].
Table 1 Chemical coupling constants for kinesins and myosin*. Motor
Motor alone (~M)
Motor.ATP (I.tM)
Motor.AMP.PNP (I.tM)
Motor.ADP.Pi (jiM)
Motor.ADP (/aM)
References
Kinesin ncd Eg5 Myosin
0.002 0.012 1 0.01-0.1
? ? ? 100
0.13 0.12 0.02 105
? 0.5f ? 50
6 6 > 10 10
[49] [49,50] [51 ] [10]
*Track dissociation constants (per actin monomer and tubulin heterodimer) are given for kinesin, ncd (a reverse-directed homologue), Eg5 (a slower homotogue) and myosin. ;BeF complex. SVery dependent on ionic strength.
Macromolecular assemblages
240
Figure 1
(a)
(b)
Weak binding nucleotide trapped
Strong binding nucteotideex~anges
Myosin S1 50kD I 20kD 1olo I 3ol0 4o01 I I 1 I I 200 500 eoo too 800 Proximal domain of motor - Distal domain of motor ~ Lever arm 25kD
0I
[
I
m:i •
•
i;
Insedion 1 A1
~'~
KoADP M-ATP M.ADP.Pi
K. . . . . K * A M ~ P N P [=ATP?] K.AD~Pi M. M.ADP
Kinesin motor d o m a i n l 1 N4 ~-,~ 295 335
m~:m~
"~ l l "
A2
IIL~ L III1~1
N1
N2
l ~ l ~i ~,2
• ::: *t3
N3 L12 I I I c~4 (~5 c~6
Insertion 2 A
l;
I ELC RLC
~,~l 340
401
I
Ncd motor domain
700
Functional and structural relationships between myosin and kinesin motors. (a) Cyclic transition of binding states between motor and track. Motor domains (K, kinesin; M, myosin) cycle between weakly bound and strongJy bound conformations, depending on whether nucleotide (solid black circle) is present or not in the active site. Both families of motor are strongly bound when the site is empty, but the transition steps between weak and strong binding differ (see Table 1). A kinesin domain remains strongly attached to tubulin whilst cleaving Pi from ATP and releasing it; following detachment kinesin becomes strongly bound again by releasing ADP. Myosin is immediately detached from actin on binding ATP and reattaches strongly by releasing Pi. Kinesin only releases ADP readily when interacting with microtubules; myosin only releases Pi readily when interacting with actin filaments (see Table 2). (b) Homology in the arrangement of sequence segments. Schematic comparison of the amino acid sequences of the $1 fragment of myosin and the motor domains of kinesin and ncd. c~-helical segments are shown in black, ~-sheet strands are shown in grey and the putative coiled-coil extensions from kinesin and ncd motor domains are shown as cross-hatched segments; the latter segments were absent from the proteins that were crystallized [1"°,2"] but were present in dimeric motors studied by electron microscopy [29"',32"]. The scale at the top shows amino acids in the myosin sequence numbered from the N terminus. The 25, 50 and 20 kDa fragments obtained by proteolysis are also marked. A1 (405-415), A2 (529-558) and A3 (626-64?) labe{ points in the myosin sequence that are believed to contact actin [16]. ELC and RLC refer to the essential and regulatory light chains of myosin II, which are associated with the long ~-helical lever arm or 'regulatory domain' (?62-842). Insertion 1 in the myosin sequence seems to be functionally equivalent to loop L8 of kinesin or ncd; insertion 2 seems to be equivalent to loop L12. Small loops at N1-N4 are responsible for binding nucleotide to kinesin or ncd; N2 and N3 are also known as 'Switch 1' and 'Switch 2' [?°°]. AMP.PNP is a nonhydrolysable analogue of ATP. Figure 1 b adapted with permission from [1"'].
Table 2 Kinetic constants for kinesins and myosin'. Motor Kinesin -MTs Kinesin + MTs ncd - Mrs ncd + MTs Eg5 - MTs Eg5 +MTs Myosin -F-actin Myosin + F-actin
Hydrolysis (s-1)
Pi release (s-1)
ADP release (s-1)
Turnover (s -1)
References
9 1O0
>10 >13 ~ ? ? ? ? 0.1 50-1 O0
0.002-0.01 295, 300 0.003 1.65 0.006 4.94 1 300-500
0.02 20, 275 0.002 1.1, 0.885 0.01 1.42, 1.925 0.05 10-1 O0
[52-55] [52-54,56] [42,56,5?] [42,56,57] [5 t] [51] [10] [10]
50-1 O0 50-100
*Rates for catalysis (two-headed motors, rates per site) are given for kinesin, ncd (a reverse-directed homologue), Eg5 (a slower homologue) and myosin. MTs, microtubules. )Equal to cycle rate. STurnover of mant-ATP (a fluorescent analogue of ATP).
Motions in homologous active sites actuate different events Mg.ATP hydrolysis within the proteins involves the cleavage of the y-phosphate bond followed by the release of phosphate (Pi) and subsequently Mg-ADP (Fig. la). The effects of the various nucleotide intermediates on track binding, and the effects of track binding on ATP turnover, differ substantially amongst the three motors (Tables 1,2). Specifically, the transitions from strong to
weak track binding occur at different points in the cycle. Myosin is unusual in that Mg-ADP.Pi is trapped in the active site in the absence of actin [11]. Actin binding releases Pi (possibly through a 'back door' [12]) and the motor locks down on to the track. Pi is rapidly released from kinesin and ned, but Mg.ADP is trapped in the absence of mierotubules [8,9]. Microtubule binding triggers Mg.ADP release and locks the motor down. In both cases, tight binding to the track is coupled to an
Structure and dynamics of molecular motors Amos and Cross
active-site conformational change that releases the trapped products and allows Mg.ATP rebinding. This ensures that nucleotide turnover is tightly coupled to track binding.
Conformational changes associated with nucleotide turnover in myosin There are good grounds for believing that Pi release triggers the major force generating conformational change in myosin [10] but attempts by electron microscopy to visualize a structural change have been repeatedly frustrated, because the weak binding myosin.Mg.ADP-Pi
941
starting state is both difficult to populate and disordered; the myosin molecules that do bind to actin seem to be
attached at many different angles. However, recent 3D image reconstruction from electron micrographs of some types of myosin S1 heads bound to F-actin [13",14 °] has revealed some domain motion coupled to Mg.ADP release. A 'lever arm' (see Fig. 2) of the myosin motor moves substantially, on the release of Mg.ADE in both brush border and smooth muscle myosin; however, no such movement appears to occur in rabbit skeletal myosin when Mg-ADP is released [15].
Figure 2 (a)
Distal domain of N motor
Lever arm
Actin contact in Insertion 2 Actin contact in Insertion 1
(b)
p,
c;, 1997 Current Opinion in Structural Biology
Comparison of crystal structures. Corresponding views of (a) myosin and (b) kinesin, looking down on the nucleotide (shown in solid black; see also Fig. 5 of [7"]). The structure of ncd (not shown) is almost identical to that of kinesin [1 °°,2°*]. The homology in the structural elements that support the nucleotide-binding pocket is less apparent than in views at right angles to this [1°°], but the myosin domains that move relative to one another [11,16] are more distinct. The distal domain (residues 45'77-762) appears to rotate around a hinge near residue 45'7 (marked by asterisk). SH1 and SH2 indicate two cysteines that can be cross-linked in solution [21,22]; their large separation in all the available crystal structures of myosin suggests that only part of the full conformational change has been shown. Insertion 1, consisting of loop L8 with strands ~5a and ~5b, and Insertion 2, consisting of loop L12, are predicted to make contact with tubulin (see Fig. 3c) [1 °°]. The crystal structure of kinesin lacks a short segment (residues 325-332) at its C terminus (shown by arrow head); the structures of this segment and the subsequent stretch of o~ helix are therefore hypothetical ['7°°].
249
Macromolecular assemblages
T h e initial evidence for relative movement of myosin domains came from fitting atomic structures into a 3D map derived by image reconstruction from electron micrographs of frozen-hydrated actin filaments complexed with myosin S1 [16]. In order to fit the crystal structures of myosin that contains bound nucleotide into the rigor structure (containing no nucleotide) imaged in the complex, it was necessary to close a cleft between two segments, referred to in Figures lb and 2 as Insertion 1 and Insertion 2. Rayment et al. [16] suggested that the initial weak binding of myosin.Mg-ADP.Pi to actin might be via a site in Insertion 2, and that this interaction might be followed by closure of the cleft and formation of an additional interaction between actin and a site in Insertion 1 (see also [17,18]). Crystal structures of the Dictyoste/ium myosin motor domain [11,19°,20 °] provide more direct evidence for nucleotide-dependent conformational changes in myosin. Two conformations have been observed: the BeF 4 and pyrophosphate complexes are thought to be analogues of a myosin.ATP transition state, whereas the AIF 4 and ADP-Vi complexes are more myosin.ADP.Pi-like. T h e transition from the ATP-like to ADP-Pi-like states partially closes the cleft between Insertion 1 and Insertion 2 (Fig. 2). This transition elicits a response in a so-called 'converter region' in the neck of the molecule. T h e crystallographic evidence is very clear, but the motions in solution may be more dramatic: two cysteines known as SH1 (Cys697) and SH2 (Cys707), which can be cross-linked in the ADP.Vi state in solution [21,22] (thereby trapping ADP.Vi in the active site), are still 18 ~ apart in the crystal structures, at opposite ends of a kinked c~ helix between the active site and the lever arm (Fig. 2a).
Lever arms as amplifiers T h e motion of a lever arm is clearly an effective way to amplify more modest motions within the myosin head [23]. Mutagenesis experiments in which extra copies of the light chain binding regions were concatenated to the natural sequence have established that the sliding velocity at pseudozero load correlates with increases in lever arm length [24]. It is not clear, however, how stiff the lever arm is. T h e replacement of the authentic lever arm with ct-actinin, which is predicted to be stiff, preserves the unloaded sliding velocity [23]. Howard and Spudich (in an appendix to [24]) considered the possibility that the lever is flexible. To what extent the lever arm functions as a genuine cantilever or as a flexible tether is unclear. Myosin light chain mediated regulation may possibly work by switching the stiffness of the lever arm. Motility assays carried out under load will be necessary to investigate these points.
Conformational changes in kinesin and ncd T h e recently solved atomic structures for kinesin and ncd [1°°,2°°] are the motor.ADP states, which are very stable
[Table 1]. No crystallographic information is available about other states, and the clearest clues to how the active sites of kinesins may move derive from work on the Ras GTPase (for reviews, see [2°°,7°°]). T h e GTPase site of Ras is homologous to the myosin and kinesin ATPases sites. For Ras and other homologous G-proteins, two small loops, named Switch 1 and Switch 2, face each other across a cleft into which the y-phosphate of the trinucleotide would protrude when present. T h e conversion from the G T P to the G D P complex induces a progressive scissor-like motion between Switch 1 and Switch 2. Vale [7 °°] has suggested that dinucleotide is subsequently released because of a subtle change in the Switch 2 region that causes the Mg2+ ion to be lost. Dinucleotide release switches kinesin into a strong binding (force-holding) conformation and presumably does so by producing some further movement around the nucleotide-binding pocket. Exactly what happcns is not clear. In contrast to the myosin situation, the connectivity between the nucleotide-binding site of kinesin or ncd and the microtubule-binding sites is only just beginning to be elucidated using mutagenesis. An ncd point mutation, on one of the [~ strands between the proposed tubulin-binding surface and the nucleotide, slows ATP turnover and affects microtubule affinity [25]. T h e 3D electron microscopy of monomeric kinesin motor domains bound to microtubules (see Fig. 3a) [26"] indicates that A D P release is coupled to an angular change in one portion of the motor domain. T h e underlying tubulin structure appears to be essentially unchanged [27], which is consistent with the observation that kinesincoated beads are still able to move on microtubules that have been cross-linked with glutaraldehyde [28]. How this relatively modest candidate power stroke might be amplified to produce the 16nm strides necessary for processivity [9,29"] is discussed below.
Interaction between kinesin and microtubules T h e definitive fitting of the atomic structures of kinesin and ncd into 3D electron microscope images of these motor domains bound to the surfaces of microtubules or tubulin sheets [29"°,30,31,32 °] will probably require images of frozen-hydrated specimens labelled with electron-dense markers. Information from crystal structures of dimeric motors, which are likely to be solved in the near future, should greatly restrict the range of possible orientations. Meanwhile, it is possible to make some tentative guesses. Figure 3b shows the kinesin crystal structure oriented so that the two putative tubulin-binding loops L8 and L12 [1*°,2*',7 °°] lie on the reverse side and, in Figure 3c, this view has been fitted to electron microscope images in two possible orientations. In both cases, the two loops are positioned on the face of kinesin that would contact tubulin and are roughly one above the other; however, the prominent spike observed on the left side of heads
Structure and dynamics of molecular motors Amos and Cross
Figure 3
243
[33-35,36"] relative to those in normal microtubules [36"',37,38]. According to consensus, features identifying the N and C termini of the tubulin monomers [33] lie in the left half of a protofilament, as viewed from outside a microtubule oriented with its plus end upwards, and close to where kinesin and ncd motor domains appear to bind (see Fig. 3a,c). Independent evidence suggests that kinesin binds near to the C termini of tubulin monomers [39,40].
The kinesin directionality problem
Fitting the kinesin crystal structure to 3D electron microscope images. (a) Part of a model combi~ning views of a microtubule decorated with monomeric kinesin motor domains in three different nucleotide states (AMP.PNP, no nucleotide [nucl], ADP; see also Fig. 3e of [27]). The individual 3D models are calculated from electron microscope images of negatively stained specimens [26°]. A prominent spike, on the left of each motor domain in this view, appears to rotate towards the microtubule plus end (oriented towards the top of the page) when Mg.ADP is lost from its nucleotide-binding site. Binding of AMP-PNP, a nonhydrolyzable analogue of ATP, does not cause a reverse movement. The vertical spacing of the motor domains corresponds to the 8 nm length of tubulin heterodimers. (b) A view of the kinesin motor domain at about 90" to that in Figure 2b, rotated so that Insertions 1 and 2 (see Figs lb,2b) are on the reverse side. In this orientation, both the N and C termini of the traceable regions of the polypeptide are towards the top of the page; there are six unseen residues at the N terminus and 24 at the C terminus. Figure redrawn from a view provided by J Kull. (c) A model obtained by combining several 3D electron microscope images of frozen-hydrated microtubules decorated with motor domains [29°°]. The longitudinal 'protofilaments' of the microtubule show a 4 nm periodicity corresponding to the tubulin monomer spacing. Attached to some sites on the protofilaments are images of monomeric kinesin (K), dimeric kinesin (attached domain, K1 ; tethered domain, K2) and dimeric ncd (N1 and N2). Superimposed on images of two kinesin monomers are miniaturised views of the kinesin crystal structure (one oriented as in Fig. 3b, the other rotated 135"). The side views on the left (based on Fig. 2b) show loops L8 (incorporating 1~5aand I]5b) and L12 binding separately to tubulin; the hypothetical dimer structure illustrates the proposal that the two strands of the coiled coil may separate to allow the tethered motor domain to find the next available binding site [8,9].
in the electron microscope images fits into two alternative features in the atomic structure. Recent reports agree about the probable orientation of tubulin protofilaments in Zn-induced 2D crystals
In 3D images of kinesin and ncd motor domains bound to tubulin [25,26",27,29,30], the two species of attached motor domains are almost indistinguishable but the second, unattached, heads of the dimeric molecules are differently oriented (Fig. 3c). The second head of a kinesin dimer, joined to the attached head at its C terminus, is oriented towards the plus end, whereas the second head of ncd, joined to its partner at the N terminus, points towards the minus end. The appropriate tilting of the free head by the bound head, as observed by electron microscopy, will clearly help to specify directionality by directionally biasing the free head. This is not in itself enough, however, because kinesin single heads are directional also. Moreover, the direction in which kinesin motors push microtubules has been shown to be unchanged by moving the tail from the C to the N terminus [41]; therefore, differences within the motor probably determine the directions of motility. What sort of differences? The main structural differences occur within a group of three small loops, L1, L5 and L9, that lie on the points of a triangle enclosing the nucleotide, and within two slightly larger loops, L2 and L l l , on either side of the y-phosphate cleft (see Figs 2b,3b). Vale and coworkers [2"°,7 °'] have suggested that the loss of y-phosphate from kinesin or ncd triggers the opposite movement of a structural unit composed of L l l and c~ helix 4 in each protein. Alternatively, Lockhart and Cross [42] have suggested that the key to directionality lies in the angle of attack of the weak binding state, in which each motor domain first attaches to tubulin (illustrated in Fig. 4). On the basis of its structural homology with myosin, we expect kinesin and ned motor domains to make their initial attachments to tubulin using just one of the two putative tubulin-binding loops. If nucleotidc hydrolysis and product release are sensed by the core structures and transmitted to a pair of tubulin-binding sites on the surface, small sequence changes within the binding sites might favour initial binding by one site rather than the other and thereby reverse the direction. In our view, this suggestion remains the most plausible candidate mechanism by which two such similar structures can develop force in opposite directions. Thus, we suspect that the initial recognition of microtubnles by the motor-Mg-ADP states of kinesin and ncd operates via different surface loops, possibly L8
244 Macromolecul assembl ar ages for kinesin and L12 for ncd. This idea should be testable using mutagenesis.
Figure4 (a)I
~
(b)
QI
site, which, quite clearly, requires some unzipping of the coiled coil via which the two motor domains associate (see Figs 3c,4a) [8]. The tethered head would then be free to diffuse randomly in search of a new binding site within the volume allowed by its tether; it will obviously be helpful if the search pattern is biased by a suitable tilting of the site to which the tether is attached. Reconciling the fixed positions of kinesin head 2 observed by electron microscopy with this prediction of mobility is a problem, but Hirose et al. [29"'] have suggested that head 2 exchanges rapidly between parked and freely diffusing states. Several groups are actively mutating the neck region of kinesin to seek an effect on processivity. There is evidence of a similar unzipping in myosin [44,45], which may allow each motor domain to search independently for a new binding site.
Synthesis: power strokes and Brownian ratchets
1997 Current Opinion in Structural Biology
Motility schemes for kinesin and ncd. (a) The processive behaviour of kinesin may possibly be explained by melting of part of the coiled-coil structure through which the motor domains (heads) are paired [8,9]. Thermal motion will then allow the tethered head to search for the nearest available binding site (stage I). One of the tubulin-binding loops binds first (stage II). After release of Mg.ADP and attachment of the second loop, the leading head is strongly attached (stage Ill). Because of the conformational change associated with ADP loss, the lead head exerts a tension on the rear head; the effect of tension is highly directional, causing the rear head (but not the leading head) to detach (stage IV) and it is pulled forwards to begin the next cycle. (b) In order to explain the different directions of kinesin and ncd movement, we suggest that initial attachment is via different loops (stage II), before settling down into the strongly attached state (stage III). In addition, on the one hand, when a kinesin head is fully attached, it needs to be pulled off much more easily by its partner pulling from the plus direction than by tension from the minus direction. On the other hand, ncd needs to resist tension from the plus direction when pulling a load towards the minus end.
The processivity mechanism of kinesin Vale et al. [43] have shown that untrapped fluoresecently labelled single (dimeric) molecules of kinesin are processive, neatly removing lingering doubts that earlier observations of processivity by optical trapping were due to the trapping scheme. Kinesin tracks microtubule protofilaments efficiently [9] and in all probability, moves 'hand over hand' in 8nm steps along a single protofilament. In order to do this, each motor domain needs to detach and swing forward 16nm to its next binding
Processive stepping by kinesin must involve a substantial diffusional component, and this concept is also influencing thoughts about the myosin mechanism [23]. We suggest that the generalized motor depicted in Figure 4a carries out a diffusional scan for its binding site using a surface loop as a lasso and subsequently locks down using a conformational change (a power stroke). The progress (step distance) is equal to the amplitude of the conformational change plus the distance diffused. The balance between the different contributions (diffusion versus power stroke) may be different between myosins and kinesins, but a role for biased diffusion in the myosin mechanism seems reasonable. The longitudinal step from one actin monomer to the next is 5.5 nm, but measurements using optical trapping experiments suggest that the shift produced may be as little as 2nm [46]. Perhaps the implausibly long lever arms in some vesicular myosins [47] imply processivity?
Dynein The study of the structure and function of dynein, another type of microtubule-associated motor, has scarcely begun compared with the progress made for the myosin and kinesin families. Kinetically, dynein appears to behave in a similar way to myosin [8]; however, a dynein molecule usually has a pair of motor domains, each at least five times the size of a kinesin motor domain, and the structural changes involved in motility are probably complex. A recent study has yielded evidence suggesting that active molecules shift by as much as 12nm during a kinetic cycle [48"].
Conclusions The elucidation of motor mechanism is one of the most challenging problems in biology, yet enormous progress has been made in the past year. Apparently, the movements made in the active sites of myosin, kinesin and ned are fundamentally homologous but each species of motor amplifies them differently. We have proposed,
Structure and dynamics of molecular motors Amos and Cross
in particular, that the two distinct tubulin-binding regions of kinesin and ncd may bind sequentially; and that the direction of morion may depend on which of the two regions binds first. We expect rapid progress in the field as this and other possible model amplification mechanisms are tested.
245
muscle myosin) about the end of the actin-binding domain, which remained unchanged. 14. •
Whittaker M, Wilson-Kubalek EM, Smith JE, Faust L, Milligan RA: A 35A movement of smooth muscle myosin on ADP release. Nature 1995, 378:748-751. See annotation [13"]. 15.
Gollub J, Creme CR, Cooke R: ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin. Nat Struct Bio/1996, 3:796-802.
Acknowledgements
16.
Molecular motors arc a large subject and the cxit~encies uf space have unfortunately prevented us citing everything and c v c u o n e we wished to. ",~e arc, however, especially gratcfut to Jon KulI f'~r pro~ iding images of the crystal structures; other ~iews of kincsin and ned can be seen at his web site [58].
Rayment I, Holden HM, Whittaker M, Yohn CB, Lorenz M, Holmes KC, Milligan RA: Structure of the actin-myosin complex and its implications for muscle contraction. Science 1993, 261:58-65.
1 "7.
Uyeda TO, Ruppel KM, Spudich JA: Enzymatic activities correlate with chimaeric substitutions at the actin-binding face of myosin. Nature 1994, 368:567-569.
18.
Ruppel KM, Spudich JA: Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft. Mol Biol Ce//1996, 7:1123-1136.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •,
of speciat interest of outstanding interest
1. •.
Kull FJ, Sablin EP, Lau R, Fletterick RJ, Vale RD: Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature 1996, 380:550-555. The atomic structure of the human kinesin motor domain is revealed to be an arrowhead-shaped domain with a few loops on its surface. The cluster of (x helices and ]3 strands forming the main domain turns out to be much more similar to the core of the myosin motor domain than was expected. 2. ..
Sablin EP, Kull FJ, Cooke R, Vale RD, Fletterick RJ: Crystalstructure of the motor domain of the kinesin-related motor ncd. Nature 1996, 380:555-559. In this paper, the crystal structure of the Drosophila motor ncd with bound Mg.ADP is compared with the kinesin structure [1°*], showing that the two motor domains are remarkably similar despite their opposite directions of movement. A model is proposed in which kinesin and ned react in opposite ways to the presence or absence of the cleavable y-phosphate of ATP.
19. •
Fisher AJ, Smith CA, Thoden JB, Smith R, Sutoh K, Holden HM, Rayment I: X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with Mg.ADP.BeF(x) and Mg.ADP.AIF4-. Biochemistry 1995, 34:8960-8972. See annotation [20"]. Smith CA, Rayment I: X-ray structure of the magnesium(ll).ADP vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. Biochemistry 1996, 35:5404-5417. These two papers [19",20"] report the crystal structures of a truncated myosin head complexed with Mg.ADP and possible phosphate analogues. The absence of most of the (x-helical neck or lever arm and its associated light chains allowed the globular motor domain to be crystallized without the modifications required for crystallization of complete $1 fragments. Structural changes observed between different nucleotide states are thus more likely to be functional. The observed response to a change in bound nucleotide is a significant rotation of the distal domain (residues 457-?62) relative to the domain that binds to actin. 20. •
21.
Burke M, Reisler E: Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin: chemical probing of movement of residues during conformational transitions. Biochemistry 1977, 16:5559-5563.
22.
Wells JA, Yount RG: Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1. Proc Nat/Acad Sci USA 1979, 75:4966-4970.
3.
Sakowicz R, Goldstein LSB: The muscle in kinesin. Nat Struct Biol 1996, 3:404-407
4.
Sellers JR: Kinesin and ned, two structural cousins of myosin. J Muse Res Cell Moti11996, 17:1 73-175.
5.
Hackney DD: Myosin and kinesin: mother and child reunited. Chem Bio11996, 3:525-528.
23.
Block SM: Fifty ways to love your lever: myosin motors. Ceil 1996, 87:151-157.
6.
Rayment I: Kinesin and myosin: molecular motors with similar engines. Structure 1996, 4:501-504.
24.
Uyeda TOP, Abramson PD, Spudich JA: The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc Nat/Acad Sci USA 1996, 93:4459-4464.
25.
Moore JD, Song H, Endow SA: A point mutation in the microtubule binding region of the ncd motor protein reduces motor velocity. EMBO J 1998, 15:3306-3314.
Vale RD: Switches, latches, and amplifiers: common themes of G proteins and molecular motors. J Ceil Biol 1996, 135:291-302. This stimulating review compares and contrasts the structures and activities of myosin, kinesin and ned with severa/G proteins whose atomic structures are known. All have a recognizably similar fold around the nucleotide, especially near the y-phosphate. Pre-existing information about the reaction of G proteins to trinucleotide hydrolysis sheds light on the probable mechanisms of motor proteins. 7. o•
Hirose K, Lockhart A, Cross RA, Amos I_A: Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. Nature 1995, 376:277-279. 3D electron microscope images of negatively stained specimens show 16protofilament microtubules from cricket sperm, decorated with monomeric kinesin motor domain with variations in bound nucleotide. A small projection from the motor domain appears to be tilted nearer to the microtubule plus end in the nucleotide-free state, as compared with the Mg.ADP-containing state. 26. •
8.
Hackney DD: The kinetic cycles of myosin, kinesin, and dynein. Annu Rev Physiol 1996, 58:731-750.
9.
Howard J: The movement of kinesin along microtubules. Annu Rev Physiol 1996, 58:703-729.
tO.
Bagshaw CR: Muscle Contraction, edn 2. Chapman Hilh New York; 1993.
27.
Amos LA, Hirose K: Microtubule-motor complexes. Curt Open Ce// Bio/1997, 9:4-11.
11.
Rayment I, Smith C: The active site of myosin. Annu Rev Physiol 1996, 58:671-702.
28.
Turner D, Chang C, Fang K, Cuomo P, Murphy D: Kinesin movement on glutaraldehyde-fixed microtubules. Anal Biochem 1996,242:20-25.
12.
Yount RG, Lawson D, Rayment I: Is myosin a 'back door' enzyme.'? Biophys J Suppl 1995, 68:44-49.
Jontes JD, Wilson-Kubalek EM, Milligan RA: A 32" tail swing in brush border myosin I on ADP release. Nature 1995, 378:751-753. These two papers, 113*,14"] compare 3D maps of F-actin decorated with myosin heads in the presence and absence of Mg-ADP. The C-terminal light chain binding domain was reported to pivot 32" (or 23" in the case of smooth 13. •
Hirose K, Lockhart A, Cross RA, Amos LA: Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules. Prec Nat/Acad Sci USA 1996, 93:9539-9544. This paper compares the two oppositely moving double-headed motors in the AMP-PNP state. The attached head of each dimer resembles singleheaded kinesin and binds to tubulin in the same way; the positions of the second heads are quite different. These are the first reported structural dif29. •,
246
Macromolecular assemblages
ferences that appear to be relevant to the opposite directions of movement of the two motors. In addition, the connections between pairs of heads allow the approximate positions of both the N and C termini to be identified in an attached head.
41.
Stewart RJ, Thaler JP, Goldstein LS: Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein. Proc Nat/Acad Sci USA 1993, 90:5209-5213.
30.
Sosa H, Milligan RA: Three dimensional structure of ncd decorated microtubules obtained by a back-projection method. J Mo/Bio/1996, 260:743-755.
42.
Lockhart A, Cross RA: Origins of reversed directionality in the ncd molecular motor. EMBO E 1994, 13:751-757.
31.
Hoenger A, Milligan RA: Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. J Mo/ Bio/1997, 265:553-564.
43.
Vale RD, Fumatsu T, Pierce DW, Romberg L, Ilarada Y, Yanagida T Direct observation of single kinesin molecules moving along microtubules. Nature 1996, 380:451-453.
44.
Offer G, Knight P: The structure of the head-tail junction of the myosin molecule. E Mo/Bio/1996, 256:407-416.
45.
Knight PJ: Dynamic behaviour of the head-tail junction of myosin. J Mo/ Bio/1996, 255:269-274.
46.
Molloy JE, Burns JE, Kenddck-Jones J, Tregear RT, White DCS: Movement and force produced by a single myosin head. Nature 1995, 378:209-212. Cope MJTV, Whisstock J, Rayment I, Kendrick-Jones J: Conservation within the myosin motor domain: implications for structure and function. Structure 1996, 4:969-987.
32. •
Arnal I, Metoz F, DeBonis S, Wade RH: Three-dimensional structure of functional motor proteins on microtubules. Curr Bio/1996, 6:1265-1270. This polarity of microtubules decorated with double-headed motors is determined in this study using microtubules assembled from centrosomes. Helically symmetrical 15-protofilament microtubules are selected from a mixed population of reassembled brain microtubules for reconstruction of 3D iraages comparing dimedc kinesin and ncd. The results agree with [29"]. 33.
Nogales E, Wolf SG, Khan IA, Luduena RF, Downing KH: Structure of tubulin at 6.5A and location of the taxol-binding site. Nature 1995, 375:424-427.
47.
34.
Ray S, Wolf SG, Howard J, Downing KH: Kinesin does not support the motility of zinc-macrotubes. Cell Motil Cytoske/ 1995, 30:146-152.
35.
Nogales E, Wolf SG, Zhan SX, Luduena RF, Downing KH: Preservation of 2-D crystals of tubulin for electron crystallography. J Struct Biol 1996, 115:199-208.
48. Burgess SA: Rigor and relaxed outer dynein arms in replicas of • cryofixed motile flagella. J Mol Biol 1995, 250:52-63. Electron microscope images of thousands of individual freeze-etched flagellar dynein arms are compared by single-particle computer analysis. When ATP is rapidly diluted out, the arms appear to be shifted 12nm away from their relaxed positions. In specimens frozen while active, a proportion of the arms are also in this maximally shifted position, suggesting it is a stage in the kinetic cycle. The remaining arms are either relaxed or in a slightly tilted conformation.
36. ••
Wolf SG, Nogales E, Kikkawa M, Gratzinger D, Hirokawa N, Downing KH: Interpreting a medium resolution model of tubulin: comparison of zinc-sheet and microtubule structure. J Mo/Biol 1996, 262:485-501. A difference map comparing unmodified Zn-tubulin with subtilisin-digested tubulin, lacking part of the C-terminal segment, shows a peak close to the rod-shaped feature tentatively identified in [33] as a predicted C-terminal c( helix. The plus-minus orientation of Zn-tubulin protofilaments was established by comparison of their projected density distribution with that of protofilaments in normal tubulin sheets of known polarity. An attempt to orient the 3D map into a helical reconstruction from ice-embedded microtubules did not lead to a single best fit, apparently because of a difference in protofilament thickness in the two assemblies. As the electron fibre diffraction patterns from microtubule bundles show a general similarity to the electron diffraction patterns from Zn-tubulin crystals, the most probable explanation is that the conformation of Zn-tubulin is simply more compact than normal tubulin. This difference may explain why motors do not interact with Zn-tubulin [34]. 3?.
Hoenger A, Milligan RA: Polarity of 2-D and 3-D maps of tubulin sheets and motor-decorated sheets. J Mol Biol 1996, 263:114-119.
38.
Hirose K, Amos WB, Lockhart A, Cross RA, Amos LA: Threedimensional cryo-electron microscopy of 16-protofilament microtubules: polarity and interaction with motor proteins. J Struct Bio1199"7, in press.
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Goldstein LSB: Structural features involved in force generation in the kinesin superfamily. Biophys J Supp11995, 68:260-266.
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Larcher JC, Boucher D, Lazereg S, Gros F, Denoulet P: Interaction of kinesin motor domains with alpha- and betatubulin subunits at a tau-independent binding site: regulation by polyglutamylation. J Bio/Chem 1996, 271:22117-22124.
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Crevei IM-T, Lockhart A, Cross RA: Weak and strong states of kinesin and ncd. J Mo/Bio/1996, 257:66-76,
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5"7.
ShimizuT, Sablin E, Vale RD, Fletterick R, Pechatnikova E, Taylor EW: Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain. Biochemistry 1995, 34:13259-13266.
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Jon Kull homepage on World Wide Web URL: http://util.ucsf.edu/people/kull/kinesin.html.