Transcript 16 - Le cytosquelette

Le cytosquelette
• I - Les principes communs aux trois types de
filaments, assemblage, désassemblage
• II - Les protéines associées et leur rôle
• III - Les moteurs moléculaires
• IV - Fonctions du cytosquelette dans la cellule
1
III - Moteurs moléculaires
Généralités
•
•
•
•
Protéines associées au cytosquelette
Se déplacent sur un filament polarisé
Cycle d'hydrolyse de l'ATP
Des douzaines
3
Spécificités
• Type de filaments pour se déplacer :
microfilaments d'actine ou microtubules
• La direction de déplacement
• La charge
– organite à membrane : Golgi, vésicules, …
– autre filament : contraction musculaire, cil,
flagelle, mitose, …
4
Analogies
• Avec :
– ADN polymérase
– ARN polymérase
– Hélicase
– Ribosomes
• Car
– utilisent de l'énergie chimique
– se déplacent sur une voie linéaire
– dans une direction qui dépend de la polarité du
support
5
Fonctionnement d'un moteur
• Une tête et une queue
• Tête = moteur
• Hydrolyse du nucléotide coordonnée avec changement de
conformation
• Cycle d'attachement / détachement
–
–
–
–
–
–
liaison au filament
changement de conformation
détachement du filament
reprise de la conformation de départ
nouvelle liaison au filament
comme une "marche à pied"
• Fonction de la tête : identité
– de la voie
– direction du mouvement
• Fonction de la queue : identité
– de la charge
– fonction biologique du moteur
6
Plan
I.
Les trois groupes de moteurs
A.
B.
C.
II.
Mode d’action
A.
B.
C.
III.
IV.
Myosines (marche sur actine)
Kinésines (marche sur tubuline)
Dynéines (marche sur tubuline)
Comment créer un mouvement ?
Transport des organites limités par une membrane
Régulation de l'activité des moteurs
Contraction musculaire
Mouvement des cils et flagelles
7
I – A - Moteurs à microfilaments
d'actine
• Myosine
• Premier moteur à être identifié dans le muscle
8
Myosine II
• Six chaînes
– Deux chaînes lourdes
– Deux fois deux chaînes légères
9
Chaînes lourdes
•
•
•
•
Tête globulaire (N terminale)
Queues longues enroulées l'une dans l'autre
Établissement de liaisons queue-queue
Fabrication de filaments épais
10
Chaînes légères
• Deux paires
11
• A - Molécule de myosine II
– deux chaînes lourdes
– quatre chaînes légères (2 fois 2)
Fig •16-51
B - Molécules de myosine en
microscopie électronique ombrées
au platine
12
Filament épais
•
•
•
•
Enroulement de deux queues
Assemblage de dimères tête bêche
Plusieurs centaines de molécules =
Un filament épais
13
Fig 16-52
• Filament épais de
myosine II
– Zone nue sans têtes
– Explique la théorie du
glissement de la
contraction musculaire
14
Myosin molecules and thick filaments
•
Fig 1 Myosin molecules and thick filaments. (a)
Striated muscle sarcomere showing overlapping
arrays of thick and thin filaments. Thick filaments are
connected at the M-line and thin filaments at the Zline. The sarcomere shortens (bringing about
contraction of muscle as a whole) by sliding of the thin
filament arrays towards each other, increasing their
overlap with thick filaments. (b) Central portion of the
thick filament model showing the helical array (red
dashed line) of pairs of myosin heads on the filament
surface (each pair is represented by a yellow sphere).
The green backbone contains myosin tails. The model
represents a filament with fourfold rotational symmetry.
The axial distance between adjacent levels of heads is
14.5 nm and, in this model, the helical repeat (43.5
nm) is three times the 14.5 nm spacing. (c) Schematic
representation of the assembly of myosin molecules in
a bipolar thick filament. The head ends of the
molecules point away from the filament center in each
half. Tails have antiparallel and parallel overlaps in the
central bare zone (free of heads), but only parallel
overlaps in the distal portions of the filament. (d)
Myosin molecule. The tail (S2 and LMM) is a coiledcoil
formed from the C-terminal halves of each heavy
chain (red and green). Heads (S1) comprise the motor
domain (MD) and light chain domain (LCD), which
contains the essential light chain (ELC, blue) and
regulatory light chain (RLC, yellow).
Curr Opin Struct Biol.
2006,16(2):204-12. Structure and
function of myosin filaments. Craig
R, Woodhead JL.
15
Organisation des têtes dans les filaments du muscle strié
•
Figure 2. Head organization in striated muscle filaments. (a) Negatively stained thick filament from tarantula
muscle (protein white). Helical tracks of heads are apparent by viewing along the filament axis at a glancing
angle. (b) Helical 3D reconstruction of tarantula thick filament (5 nm resolution) based on negative-stain
images. Data set from surface rendered using UCSF Chimera . (c) Single-particle 3D reconstruction of
tarantula thick filament (2.5 nm resolution) based on cryo-EM images . The axial distance between adjacent
levels of heads is 14.5 nm and the helical repeat is 43.5 nm. The ‘tilted-J’ (blue dots) represents a pair of
myosin heads. The larger diameter of this reconstruction compared with (b), which is at the same scale, is
due to shrinkage with negative staining (the heads also appear to partially collapse onto the backbone,
resulting in loss of information and lower resolution in negative stain). (d) Reconstruction in (c) fitted with the
atomic model of smooth muscle myosin (PDB code 1i84) at two 14.5 nm levels . In both the atomic model
(based on crystallographic studies) and the filament, one head of each pair (green) has its actin-binding site
blocked by binding to the converter region (located in the motor domain) and essential light chain of the other
(red). Interactions also occur between heads from different levels. S2, whose position was uncertain in the
atomic model, has been modeled as an α-helical coiled-coil (the α-helices have the same colors as the
corresponding heads from which they arise), filling a rod of density in the reconstruction that runs from the
head–head junction down towards the filament backbone. S2 appears to interact with blocked heads from
both its own and the next 14.5 nm level. These multiple interactions could prevent the heads from hydrolyzing
ATP and interacting with actin, thus switching the filament off.
Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function
of myosin filaments. Craig R, Woodhead JL.
16
Structure des filaments épais du muscle strié
•
Figure 3. Backbone structure of striated muscle thick filaments (transverse views, shown for a filament with fourfold rotational
symmetry). (a) Molecular crystal model. Each circle represents one myosin tail). (b) Subfilament model. Each subfilament is
4 nm in diameter and contains three myosin tails.. (c) Projected density of a 43.5 nm repeat of tarantula thick filament
reconstruction (protein white). Twelve subunits, 4 nm in diameter and of high density (red circles), representing the thick
filament backbone, run parallel to the filament axis — as predicted by the subfilament model for a filament with the tarantula
symmetry. Note that (c) is shown at a smaller scale than (a,b); lower density material at higher radius represents heads.
Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function
of myosin filaments. Craig R, Woodhead JL.
17
Protéines des filaments épais du
muscle strié des vertébrés en
dehors de la myosine
•
Figure 4. Non-myosin proteins in vertebrate striated muscle thick filaments. Schematic diagram of a sarcomere (thin filaments
omitted) showing a thick filament (green backbone, myosin heads omitted) and myosin-associated proteins. Titin molecules
(blue) run from the M-line (where they overlap with molecules from the opposite half of the sarcomere), along the thick filament,
then through the I-band (the zone between the thick filament and the Z-line), where they have an elastic domain, and ending at
the Z-line, where they would overlap with titin from the next sarcomere. Myosin-binding proteins (MyBP, yellow), primarily MyBPC, occupy 11 sites 43 nm apart in the proximal portions of each half thick filament.
Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function
of myosin filaments. Craig R, Woodhead JL.
18
Schéma d’un filament épais à
polarité latérale (muscle lisse)
•
Figure 5. Schematic diagram of a side-polar thick filament. In the side-polar mode of assembly, found in smooth muscle thick
filaments, antiparallel tail interactions occur along the entire length of the filament (c.f. bipolar filaments), so that opposite sides,
rather than opposite ends, of the filament have opposite polarity. Functionally, the filament is still bipolar and able to pull actin
filaments in opposite directions from opposite ends. The double arrows indicate how bipolar myosin dimers could easily
associate with and dissociate from the filament ends, consistent with assembly-disassembly processes thought to occur in some
smooth muscles in different physiological states.
Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function
of myosin filaments. Craig R, Woodhead JL.
19
Domaines de queues de myosine
impliqués dans l’assemblage des
filaments épais
•
Figure 6. Myosin tail domains involved in thick filament assembly. Schematic representation of the myosin tail sequence
showing the locations of various regions thought to be involved in the correct assembly of thick filaments (composite diagram
based on different myosins; not all sites are involved in all myosins). These include a negative charge cluster, N1 (red), and two
positive charge clusters, P1 (blue) and P2 (purple); an assembly competence domain or ACD (yellow) [55 and 58••]; the nonhelical tailpiece or NHT (green); and four skip residues (dark blue). Smooth muscle and non-muscle myosins lack the indicated
skip residue.
Curr Opin Struct Biol. 2006,16(2):204-12. Structure and function
of myosin filaments. Craig R, Woodhead JL.
20
Théorie du glissement
• Muscle squelettique
– Orientations opposées des têtes de myosine dans
le filament épais
– Permet le glissement de filaments d’actine
d’orientation opposée
• Cœur et muscle lisse
– Même disposition mais gènes différents
21
Digestions enzymatiques
• Chymotrypsine et papaïne
– Séparation de la tête appelée S1
– S1 est responsable du glissement
22
Démonstration de l’activité motrice des têtes de myosine
2 filaments
d'actine
marqués en
vert et en
rouge
Filament d'actine marqué
Vue toutes les 0,6 sec
Vitesse  4 m/sec
Fig 16-53
S1
23
Les myosines
• La myosine n'est pas que musculaire
• Dans l’amibe Acanthamoaba castellanii, myosine
– même moteur
– queue différente
– monomère  myosine I 
• Myosine musculaire renommée myosine II (deux
têtes)
• Actuellement ordre de découverte ( XVIII)
• Puis nombreuses autres myosines
– moteur reconnaissable en -N
– queue variable en -C
– fonctions inconnues pour la plupart
24
• Arbre de la
superfamille
des myosines
Fig 16-54(A)
– Myosine I et II
les plus
répandus
– Myosine XIV
Toxoplasma et
Plasmodium
25
Structure des chaînes lourdes de myosine
que chez les plantes
que chez les
vertébrés
Fig 16-54(B)
que chez les plantes
26
Chaînes lourdes de myosine
• Forment dimères ou monomères (I, IX, XIV)
• Myosine VI : marche vers le – (la seule) de
l ’actine (insertion dans le domaine de la tête)
27
Chaînes lourdes de myosine
• Saccharomyces cerevisiae : deux myosines I,
une myosine II, deux myosines V
• Caenorhabditis elegans : 15 gènes de
myosine (7 classes)
• Homme : au moins 40 gènes
28
Rôle des myosines
• Myosine II
– Contraction musculaire et non musculaire
– Cytodiérèse
– Migration cellulaire
• Myosine I
– second site de liaison à l’actine ou site de liaison à une
membrane
– organisation intra cellulaire
– expansions cellulaires
• Myosine V
– transport des vésicules et des organites
• Myosine VII
– oreille interne
29
I – B & C - Moteurs à microtubules
B. Kinésines (superfamille)
– la kinésine
– les kinesin-related proteins (KRPs) au moins 10
familles de KRPs
C. Dynéines
30
B - La kinésine
• Moteur protéique
• Se déplace sur les microtubules
• Axone géant de calmar (transport d’organites
à membrane)
• Transport dans le sens +
• Semblable à la myosine II
– deux chaînes lourdes et deux chaînes légères
– dimérisation
• Saccharomyces cerevisiae : 6 kinésines
• Caenorhabditis elegans : 16 kinésines
• Homme :  40 kinésines
31
• Kinésines et
Kinesin-related
proteins
– Kinésine
conventionnelle
Fig 16-55
• moteur Nterminal
• queue Cterminale :
transport
d'organites à
membrane
32
• Kinésines et
Kinesinrelated
proteins
– KIFC2
Fig 16-55
• moteur Cterminal
• comprend
– protéine Ncd
de la
drosophile
– protéine
KAR3 de la
levure
• se dirige dans le
sens -
33
• Kinésines et
Kinesinrelated
proteins
– KIF2
Fig 16-55
• moteur au
milieu de la
chaîne lourde
• pas d'activité
moteur
classique
• se lie aux
extrémités des
microtubules
pour augmenter
leur instabilité
dynamique 
nom de
catastrophine 34
• Kinésines et
Kinesinrelated
proteins
– KIF IB
Fig 16-55
• agit comme
monomère
• déplace les
vésicules le
long des
microtubules
35
BimC (KRP)
• BimC
– forme des tétramères
– s'associent par les queues  moteur bipolaire 
fait glisser les microtubules les uns sur les autres
36
http://www.proweb.org/kinesin/KinesinTree.html
37
Microtubule protofilament with the bound motor domain of Neurospora crassa conventional kinesin
• Microtubule protofilament with the bound motor domain of Neurospora
crassa conventional kinesin. Model based on Song et al. (2001). In the
microtubule, two alpha-tubulin subunits (alphaT1 and alphaT2) and a
beta-tubulin (betaT1) are shown (structure adapted from Lowe et al.,
2001). Kinesin would move along the microtubule towards the top of the
figure. N. crassa kinesin and the microtubule interact predominantly
through helices H11 and H12 (yellow coils) and the carboxy-terminal tail
(thin red strand, C) of betaT1, and the region of kinesin that includes the
structural elements in dark blue (alpha4-L12-alpha5 and beta5a-L8).
Other interactions may include the carboxy-terminal tail (pink, C) of
alphaT2 and kinesin’s neck linker/neck region (grey), as well as alphaT1
and kinesin’s L11. The top of the kinesin core shows the neck domain in
two conformations: a random coil found in NcKin (protein backbone in
grey), and the ordered conformation of alpha7 from rat brain kinesin
(RnKin; Sack et al. 1997) in red. The group of yellow spheres represents
GTP and GDP in tubulin and ADP in NcKin. The drug taxol (red spheres)
is shown bound to betaT1
38
C - Les dynéines
•
•
•
•
Moteur protéique
Se déplacent sur les microtubules
Transport dans le sens Sans rapport avec la super famille des
kinésines
• Deux ou trois chaînes lourdes associées à de
nombreuses chaînes légères
• Deux catégories
– ancienne : cytoplasmique
– plus récente : axonémale
39
Fig 16-56
25 nm
•
•
•
•
Homodimère
Deux têtes moteurs
Euk +++
Vésicules, Golgi
• Hétéro et homodimères
• Deux ou trois têtes moteur
• Mouvements rapides (cils et
flagelles)
40
Dynéines / kinésines
Dynéines
Kinésines
•
•
•
•
•
•
•
•
Direction –
Très gros
Très rapides
14 m/sec
Direction +
Plus petites
Plus lentes
Les plus rapides : 2-3 m/sec
41
Analogie myosine kinésine
• Moteur myosine > moteur kinésine (850 vs
350  )
• Myosine marche sur actine / kinésine sur MT
• Pas de séquences d' en commun
• Mais moteurs presque identiques en 3-D
• Les différences viennent des boucles qui
s'échappent du noyau central
– site de liaison à l'actine (pour la myosine)
– site de liaison aux microtubules (pour la kinésine)
42
• Analogie
myosine
kinésine
Fig 16-57
43
Rôle du noyau central dans la
génération de force
Analogie avec le site de
liaison nucléotidique
des petites GTPases
de la superfamille Ras
– conformations différentes :
GTP-lié actif / GDP-lié inactif
44
•
•
•
•
Amos,LA1997
En haut à gauche : Kinesin and ncd bind to the same site on tubulin. Tubulin sheets decorated with motor domains and then negatively stained show an 8 nm
repeating polar pattern (much magnified in [a], compared with in [b–e]). The pattern is shown as it appears when microtubule plus ends are oriented towards the top
of the page. (a) shows an image of tubulin decorated with ncd that was obtained by computer analysis of many sheets. Solid contours represent higher protein
density; and dashed contours include more stain and thus less protein. Tubulin sheets decorated with kinesin heads give an extremely similar image (not shown).
(b–e) show individual decorated sheets with visible ends (top of each part of the figure). Protein appears white here; surrounding stain is black. (b,d) show
unprocessed images; (c,e) show the sheets after computer enhancement of the periodic signal and filtering out of noise. Whether the decoration is with kinesin or
ncd, the pattern stops at the same stage at the end of the sheet, suggesting that kinesin and ncd bind in the same way to a- and b-tubulin.
En haut à droite : Various polymers of tubulin. All the tubulin polymers that have been used in electron microscopic studies of tubulin–motor-domain complexes
consist of polar arrangements of longitudinal protofilaments. Each protofilament is a linear arrangement of ab-tubulin heterodimers (each being shown here as a light
monomer above a dark monomer). Most natural microtubules have 13 protofilaments but those assembled in vitro vary in protofilament number. (a) A tubulin sheet,
or opened-out microtubule. Purified tubulin assembles mainly into the so-called B-lattice, in which heterodimers in adjacent protofilaments line up at a shallow angle.
(b) A reassembled microtubule with 10 protofilaments. If the heterodimers lie on a single shallow helix, as shown here (see the laterally exposed dimers at the top
and bottom of the tube), the subunit is helically symmetrical; except near the broken ends, every dimer has an identical set of neighbours. (c) A 14-protofilament
microtubule exhibiting a ‘seam’ where lateral contacts are between light and dark subunits rather than between like subunits. (Note also the ends of 1.5 dimer
helices at the top and bottom of the tube.) Reassembled 13- or 14-protofilament microtubules must always have at least one such seam; those with any other
number of protofilaments may also have seams. (d) Natural 16-protofilament microtubules are apparently always seamless and, therefore, helically symmetrical,
with two shallow dimer helices. When there are 13 protofilaments in a microtubule (not shown) they are exactly longitudinal, but other numbers of protofilaments
twist slowly around their tubes.
En bas à gauche : Comparison of different 3-D images of tubulin–motor-protein complexes. (a) Set of 3-D images obtained by combining tilted views of negatively
stained tubulin sheets. The left-hand protofilament is undecorated; the two monomers of a tubulin heterodimer are labelled (T). The middle and right-hand
protofilaments are decorated with monomeric kinesin (K) and ncd (N), respectively. (b) Merged 3-D images of frozen, hydrated 16-protofilament microtubules. Part of
an undecorated microtubule is shown at the bottom, with tubulin monomers labelled ‘T’; successive helical segments are decorated with monomeric kinesin (K),
dimeric kinesin (attached head, K1; tethered head, K2) and dimeric ncd (attached head, N1; tethered head, N2). (c,d) Images of frozen, hydrated 10-protofilament
microtubules The models have been inverted so that microtubule polarity is consistent with (a) and (b). (c) shows an undecorated microtubule with tubulin monomers
labelled ‘T’; (d) shows a microtubule decorated with monomeric kinesin (K). (e) Merged 3-D images of negatively stained 16-protofilament microtubules. Successive
helical turns show microtubules decorated with monomeric kinesin under different nucleotide conditions. White lines on the right-hand side indicate the change in tilt
of a projecting feature, from roughly horizontal (ADP-bound state) to 45° (nucleotide-free [no nucl] and AMP.PNP-occupied states).
En bas à droite : Structural interactions between tubulin (grey circles) and motor domains (white shapes). On the left is an enlargement of part of the decorated
tubulin sheet on the right. a, a-tubulin; b, b-tubulin. Tubulin dimers appear to be oriented so that the b-tubulin monomer (shown here as a light grey subunit) is
located towards the microtubule plus end [44,45·]. Image reconstructions (see Fig. 3) show that both kinesin and ncd motor domains contact two tubulin subunits;
this is consistent with findings by cross-linking and blot overlay that both motors interact with both tubulin species. As there are no extra, undecorated tubulin
monomers at the ends of the decorated tubulin sheets (see Fig. 1), the two tubulin monomers associated with one motor head domain are most probably a stable
heterodimer. The underlying shapes of the two sorts of monomer in Figure 1a, as compared with undecorated a- and b-tubulin, also support this positioning of the
motor domains relative to tubulin dimers. The points at which double heads of kinesin or ncd are joined (see Fig. 3b) indicate the approximate locations of the amino
(N) and carboxyl (C) termini of the motor domains.
45
•
En haut à gauche Comparison of crystal structures.
–
•
Amos,LiA1997
En haut à droite Fitting the kinesin crystal structure to 3D electron microscope images.
–
–
–
•
(a) Part of a model combining views of a microtubule decorated with monomeric kinesin motor domains in three different nucleotide states (AMP.PNP, no nucleotide [nucl], ADP. The
individual 3D models are calculated from electron microscope images of negatively stained specimens. 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 1b,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.
(c) A model obtained by combining several 3D electron microscope images of frozen-hydrated microtubules decorated with motor domains. 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 b5a and b5b) 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.
En bas à droite Functional and structural relationships between myosin and kinesin motors.
•
•
Corresponding views of (a) myosin and (b) kinesin, looking down on the nucleotide (shown in solid black). The structure of ncd (not shown) is almost identical to that of kinesin. The
homology in the structural elements that support the nucleotide-binding pocket is less apparent than in views at right angles to this, but the myosin domains that move relative to one
another are more distinct. The distal domain (residues 457–762) appears to rotate around a hinge near residue 457 (marked by asterisk). SH1 and SH2 indicate two cysteines that
can be cross-linked in solution; 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 b5a and b5b, and Insertion 2, consisting of loop L12, are predicted to make contact with tubulin (see Fig. 3c). 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 a helix are therefore
hypothetical.
Cyclic transition of binding states between motor and track. Motor domains (K, kinesin; M, myosin) cycle between weakly bound and strongly 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 S1 fragment of myosin and the motor domains of kinesin and ncd. a-helical segments are shown in black, b-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 but were present in dimeric motors studied by electron microscopy. 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 (626–647) label points in the myosin sequence that are believed to
contact actin. ELC and RLC refer to the essential and regulatory light chains of myosin II, which are associated with the long a-helical lever arm or ‘regulatory domain’ (762–842).
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’ [7··]. AMP.PNP is a nonhydrolysable analogue of ATP.
En bas à gauche 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 III). 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.
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