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Motor Proteins

Molecular motors use adenosine triphosphate (ATP) hydrolysis to power movements of subcellular com­ ponents, such as organelles and chromosomes, along the two polarized cytoskeletal fibers: actin filaments and microtubules. No motors are known to move on intermediate filaments. Motor proteins also produce force locally within the network of cytoskeletal poly­ mers, which transmits these forces to determine the shape of each cell and, ultimately, the architecture of tissues and whole organisms. Chapters 37 to 39 and 44 illustrate how motors move cells and their internal parts. Just three families of motor proteins—myosin, kinesin, and dynein—power most eukaryotic cellular movements (Fig. 36.1 and Table 36.1). During evolution, myosin, kinesin, and Ras family guanosine triphospha­ tases (GTPases) appear to have shared a common ances­ tor (Fig. 36.1), whereas dynein is a member of the AAA adenosine triphosphatase (ATPase) family (Box 36.1). Although the ancestral genes appeared in prokary­ otes, and prokaryotes have homologs of both actin and tubulin, none of these motor proteins has been found in prokaryotes. Over time, gene duplication and diver­ gence in eukaryotes gave rise to multiple genes for myosin, dynein, and kinesin, each encoding proteins

C H A P T E R

36

with specialized functions. Even the slimmed down genome of budding yeast includes genes for five myosins, six kinesins, and one dynein. Table 36.1 lists other protein machines that produce molecular movements during protein and nucleic acid synthesis, proton pumping, and bacterial motility. Motor proteins have two parts: a motor domain that uses ATP hydrolysis to produce movements and a tail that allows the motors to self­associate and/or to bind particular cargo. Within the three families, the tails are more diverse than the motor domains, allowing for spe­ cialized functions of each motor isoform. All motor proteins are enzymes that convert chemical energy stored in ATP into molecular motion to produce force upon an associated cytoskeletal polymer (Fig. 36.2). If the motor is anchored, the polymer may move. If the polymer is anchored, the motor and any attached cargo may move. If both are anchored, the force stretches elastic elements in the molecules transiently, but nothing moves, and the energy is lost as heat. Cells use all these options (see Chapters 37 to 39). Biochemists originally discovered and purified these motors using enzyme (eg, ATP hydrolysis) or in vitro motility assays (Fig. 36.11). With the prototype motors identified, investigators found further examples and

Primodial NTPase Primodial GTPase Primodial myosin Primodial kinesin Primodial AAA ATPase Many Many Many Dyneins
Primodial NTPase
Primodial GTPase
Primodial myosin
Primodial kinesin
Primodial AAA ATPase
Many
Many
Many
Dyneins
Other AAA-
contemporary
contemporary
contemporary
ATPases
GTPases
myosins
kinesins

FIGURE 36.1   Evolution   of   myosin,   kinesin,  and   dynein  adenosine  triphosphatase   (ATPase)   motors   from   genes   that   encoded   two   primordial   proteins   that   bound   and   hydrolyzed   nucleoside   triphosphates.   Gene  duplication   and  divergence  created  genes   for   many   contemporary   motors.  

623

624 SECTION IX n Cytoskeleton and Cellular Motility

TABLE 36.1

Mechanochemical Enzymes and Other Proteins That Produce Movements

 

Families

Track

Direction

Cargo

Energy

ATPases

Myosins

Muscle myosin

Actin

Barbed end

Myosin filament

ATP

Myosin II

Actin

Barbed end

Myosin, actin

ATP

Myosin I

Actin

Barbed end

Membranes

ATP

Myosin V

Actin

Barbed end

Organelles

ATP

Myosin VI

Actin

Pointed end

Endocytic vesicles

ATP

Dyneins

Axonemal

Microtubule

Minus end

Microtubules

ATP

Cytoplasmic

Microtubule

Minus end

Membranes, chromosomes

ATP

Kinesins

Kinesin­1

Microtubule

Plus end

Membranes, intermediate filaments

ATP

Kinesin­14

Microtubule

Minus end

? Microtubules

ATP

Other Mechanochemical Systems

 

Polymerases and Helicases

Ribosome

mRNA

5to 3

None

GTP

DNA polymerase

DNA

5to 3

None

ATP

RNA polymerase

DNA

5to 3

None

ATP

CMG DNA helicase

DNA

DNA

ATP

RNA helicases

RNA

RNA

ATP

Conformational System

Spasmin/centrin

None

None

Cell, basal body

Ca 2 +

Polymerizing Systems

Actin filaments

None

Barbed end

Membranes

ATP

Microtubules

None

Plus end

Chromosomes

GTP

Worm sperm MSP

None

Not polar

Cytoskeleton

Rotary Motors

Bacterial flagella

None

Bidirectional

Cell

H + or Na + gradient

F­type ATPase

None

Bidirectional

None

H + or ATP

V­type ATPase pump

None

None

ATP

ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; GTP, guanosine triphosphate; mRNA, messenger RNA; MSP, major sperm protein. The terms “barbed” and “pointed” end refer to the appearance of actin filaments decorated with a myosin fragment (Fig. 33.8).

variant isoforms of each by purifying proteins, cloning complementary DNAs (cDNAs), sequencing genomes, or genetic screening.

Myosins

Myosins are the only motors that are known to use actin filaments as tracks. Members of the diverse myosin superfamily arose from a common ancestor and share a motor unit called a myosin “head” that produces force on actin filaments (Fig. 36.3). One or two heads are attached to various types of tails that are adapted for diverse purposes, including polymerization into filaments, binding membranes, and interacting with various cargos. Myosin heads consist of two parts. A catalytic domain at the N­terminus of the myosin heavy chain binds and hydrolyzes ATP and interacts with actin filaments. Light chain domains consist of an α ­helical extension of the

heavy chain from the catalytic domain associated with one to seven light chains. Light chains are related to calmodulin (see Fig. 3.12), which also serves as a light chain for many myosins.

Myosin Mechanochemistry

Myosin was discovered in skeletal muscle and used to

establish general principles that apply, with interesting variations, to energy transduction by all myosins. Muscle myosin is responsible for the forceful contraction of skeletal muscle (see Chapter 39). Like other types of myosin­II, it has two heads on a long tail formed from an

α ­helical coiled­coil. These tails polymerize into bipolar

filaments (see Figs. 5.7 and 39.6). The head of muscle myosin was originally isolated as a proteolytic fragment called subfragment­1 (Fig. 36.3). The N­terminal 710 residues of the heavy chain form the globular catalytic domain. The nucleotide binding site in the core of the catalytic domain is formed by a β ­sheet

Generic motor with stretched spring

Force Cytoskeletal fiber Motor ATP Spring ADP + P i Force Support or cargo Resulting
Force
Cytoskeletal
fiber
Motor
ATP
Spring
ADP + P i
Force
Support or
cargo
Resulting movement with anchored motor
Fiber moves
Support
Resulting movement with anchored fiber
Motor and cargo move
Cargo
Result with anchored fiber and anchored motor
Force
Force
Support

Spring stretched, force transmitted through fiber to anchoring sites, no movement, energy lost as heat

FIGURE 36.2GENERAL FEATURES OF ATPase MOTORS.   Motors bind  stably  to  a  support  or  cargo  and  transiently  to  a  cytoskel - etal  fiber  (actin  filament or microtubule).  Energy  liberated  by  adenosine  triphosphate  (ATP)   hydrolysis  produces  force   to   stretch   an   elastic   element  somewhere  in  the  physical  connection between  the  cargo  and  the   cytoskeletal  fiber.   The   resulting   motion  depends   on  whether   the   force  in   the   spring   exceeds   the   resistance   of   the   fiber   or   the   cargo. 

flanked by α ­helices with a topology similar to Ras GTPases (see Fig. 4.6) despite little sequence similarity. The γ­phosphate of ATP inserts deeply into the nucleotide­ binding site with the adenine exposed on the surface. Actin binds more than 4 nm away from the nucleotide

on the other side of the head. A region of the heavy chain called the converter subdomain is attached to the light- chain domain composed of an essential light chain and

a regulatory light chain wrapped around and stabilizing

a long α ­helix formed by the heavy chain. The inter­ action of light chains with the heavy chain α ­helix

CHAPTER 36 n Motor Proteins

625

BOX 36.1

AAA Adenosine Triphosphatases

The common ancestor of life on the earth had a gene for a versatile adenosine triphosphate (ATP)­binding domain. Through gene duplication and divergence this progenitor gave rise to the AAA family of adenosine triphosphatases (ATPases) in all branches of the phylogenic tree. Given the remarkable variety of functions of the contemporary proteins, the name “ATPases Associated with Diverse Activities” is apt. The family now includes regulatory subunits of proteasomes (see Fig. 23.8); proteases from prokaryotes, chloroplasts, and mitochondria; Hsp100 protein folding chaperones; dynein microtubule motors (Fig. 36.14); the microtubule severing protein katanin (see Fig. 34.8); activators of origins of replication (including ORC1, 4, and 5 and Mcm­7 [see Fig. 42.8]); clamp loader proteins for DNA polymerase processivity factors (see Fig. 42.12); two proteins required for peroxisome bio­ genesis (see Table 18.1); and proteins involved in vesicular traffic such as NSF (the N­ethylmaleimide­sensitive factor [see Fig. 21.15]). AAA domains have a common fold with a catalytic site that binds and hydrolyzes ATP. A “Walker A” motif of conserved residues interacts with the β ­ and γ ­phosphates of ATP, and “Walker B” motif residues participate in ATP hydrolysis. Many AAA ATPases form ring­shaped hexamers of identical subunits or up to six different AAA subunits although the dynein heavy chain has six AAA domains in one large polypeptide. Often, an arginine residue from the adjacent subunit in the hexamer inserts into the active site and facilitates conformational changes in response to ATP binding and release of the γ­phosphate.

A B Actin-binding Actin-binding site site Active Active site site ELC ELC RLC RLC
A
B
Actin-binding
Actin-binding
site
site
Active
Active
site
site
ELC
ELC
RLC
RLC

FIGURE 36.3 ATOMIC STRUCTURE OF THE HEAD OF MUSCLE MYOSIN.A,  Ribbon   drawing   of   the   polypeptide  back- bones.   B,  Space-filling   model.   Heavy  chain   residues   4–204   (green);  heavy  chain   residues   216–626   (red);  heavy  chain   residues   647–843   (purple):  essential   light   chain   (ELC   [yellow]);  regulatory  light   chain   (RLC   [orange]).  The  myosin   light  chains   consist   of   two   globular  domains   connected   by   an   α -helix,  like   calmodulin   and  troponin   C.   (For   refer- ence,   see  Protein   Data   Bank   [PDB;   www.rcsb.org ]  file2MYS .)

626 SECTION IX n Cytoskeleton and Cellular Motility

Beginning Pointed end A B C D of stroke Catalytic domain End of stroke
Beginning
Pointed end
A
B
C
D
of stroke
Catalytic
domain
End of
stroke

WITH MYOSIN HEADS. A,   Electron   micrograph   of   frozen-hydrated  actin   filaments   fully  

occupied  with   myosin   heads.   B,  Three-dimensional   reconstruction   from   electron   micrographs   of   an   actin   filament   saturated   with   myosin   heads.   C,  Superimposition  of  atomic   models   of  the   actin  filament   and  one   myosin   head   on   the   reconstruction   of   the  decorated   filament   ( blue  cage-like   surface).   D,  Space-filling  atomic   model   of   an   actin   filament  with   one   attached   muscle  myosin   head  showing   the   light-chain   domain   in   two   posi - tions:  (1)  the   end   of   the   power   stroke   as   observed   in   the   absence  of   ATP  (blue),  and  (2)   the  postulated  beginning  of   the   power   stroke   (pink)  deduced  from   X-ray   structures   of   isolated   heads  and   spectroscopic   studies.   The  catalytic  domain   (red)  is  fixed  in   one   position   on   actin   (yellow).

(Courtesy  R.   Milligan,   Scripps  Research   Institute,   La   Jolla,   CA.)

FIGURE 36.4

ACTIN FILAMENTS DECORATED

resembles calmodulin binding to its target proteins (see Fig. 3.12). Myosin heads bind tightly and rigidly to actin fila­ ments in the absence of ATP. This is called a rigor complex, because it forms in muscle during rigor mortis when ATP is depleted after death. Myosin heads bound along an actin filament form a polarized structure, resem­ bling a series of arrowheads when viewed from the side (Fig. 36.4). The heads bind at an angle and wrap around the filament. Their orientation defines the barbed and pointed ends of the actin filament (see Fig. 33.8). All known myosins, except myosin­VI, move toward the barbed end of the filament. The atomic structures of the myosin head and actin filament fit nicely into the three­dimensional struc­ ture of the decorated filament determined by electron microscopy, providing the structural starting point for understanding the mechanics of force production (Fig. 36.4). Each myosin head contacts two adjacent actin subunits.

Actomyosin Adenosine Triphosphatase Cycle

Myosin uses energy from ATP hydrolysis to move actin filaments, so an appreciation of the mechanism requires an understanding of the biochemical steps along the reaction pathway. Fig. 36.5A looks intimidating, but working through it one step at a time reveals its logic and simplicity. Note that the mechanism consists of two parallel lines of chemical intermediates. First, consider the bottom line showing the reactions that explain why myosin alone turns over ATP remarkably slowly, at a rate of only approximately 0.02 s 1 :

Step 1. At physiological concentrations of ATP, myosin binds ATP in less than 1 millisecond. Energy from ATP binding allows a conformational change in the myosin

that can be detected by a change in the intrinsic fluo­ rescence of the protein itself. Step 2. The enzyme catalyzes the hydrolysis of ATP. This reaction is moderately fast (>100 s 1 ) and readily revers­ ible. The equilibrium constant for hydrolysis on the enzyme is near 1, so each ATP is hydrolyzed to adenos­ ine diphosphate (ADP) and inorganic phosphate and the triphosphate is resynthesized several times before the products dissociate from the enzyme. ATP splitting provides energy for a second conformational change, reflected in a further increase in the fluorescence of the myosin. These conformational changes reorient the converter subdomain and the light chain domain poised to undergo the molecular rearrangements that subsequently produce movement. Step 3. Inorganic phosphate (P) slowly dissociates from the active site (at a rate of approximately 0.02 s 1 ) by escaping through a narrow “back door” on the far side of the enzyme. This is the rate­limiting step in the pathway. The loss of phosphate is coupled to confor­ mational changes that return myosin toward its basal state. The phosphate dissociation step has the largest negative free energy change, so it is presumed that energy derived from ATP binding and hydrolysis and stored in conformational changes in the myosin head is used to do work or dissipated as heat at this point in the reaction pathway. Step 4. Once phosphate dissociates, ADP leaves rapidly from the “front door.”

To summarize, in the absence of actin filaments, ATP binds rapidly to myosin and is rapidly but reversibly split, and the products slowly dissociate from the active site. The overall cycle of the enzyme is limited by the slow conformational change coupled to phosphate dissocia­ tion. Energy derived from ATP binding and hydrolysis is

A

CHAPTER 36 n Motor Proteins

Strong Weak Strong A–M A–M*T A–M**DP A–MD A–M 1 ′ 2 ′ 3 ′ 4
Strong
Weak
Strong
A–M
A–M*T
A–M**DP
A–MD
A–M
1 ′
2
3
4 ′
1 2
3
4
M
M*T
M**DP
MD
M

Myosin bound to actin

Free myosin

≥ 1000 s -1 100 s -1 10 s -1 1s -1 0.1 s -1
≥ 1000 s -1
100 s -1
10 s -1
1s -1
0.1 s -1

627

B P ADP ATP ADP ADP B + Pi + Pi
B P
ADP
ATP
ADP
ADP
B
+ Pi
+ Pi
Pi Rapid equilibrium free and bound Phosphate ADP dissociates ATP binding dissociates Head dissociates Light
Pi
Rapid equilibrium
free and bound
Phosphate
ADP dissociates
ATP binding
dissociates
Head dissociates
Light chain
domain rotates
ATP hydrolysis
FIGURE 36.5  
MYOSIN ATPase
MECHANISMS.   A,   A   diagram   of   the   actomyosin   ATPase   cycle   of   striated   muscle   myosin-II   showing   the  

actin   filament   (A),   myosin   head  (M),   ATP   (T),   adenosine   diphosphate   (ADP)  (D),   and   inorganic   phosphate   (P).   Transient-state  kinetics  revealed   the major  chemical  intermediates  and   the   rate   constants   for   their   transitions.  Arrow sizes  are   proportional   to   the  rates   of   the   reactions,   with  second- order   reactions   adjusted   for   physiological   concentrations   of   reactants.   One  or   two asterisks  indicate   conformational   changes   in   the   myosin   head   induced  by   ATP   binding   and   hydrolysis.   Myosin   without   nucleotide   (M)  and  myosin   with  ADP   (MD)  bind  much   more  tightly   to   actin   filaments  than   do   AMT   and   AMDP.  The   weakly   bound   AMT   and   AMDP   intermediates   are  in   a  rapid   equilibrium  with   free  MT   and   MDP.   The   beige  shading  shows  the   main   pathway  through   the   reaction.  B,   The   postulated   force-producing   structural   changes   in   the   orientation   of   the   light-chain   domain   (purple  and   blue)   coupled   to   the   myosin   ATPase  cycle.   (B,  Data   from   R.   Vale,   University  of   Califor nia,   San   Francisco,   and  R.  Milligan,   Scripps   Research   Institute,   La   Jolla,   CA.)

used for a conformational change in the myosin head that is dissipated when phosphate dissociates. The upper line in Fig. 36.5A shows myosin associated with an actin filament. The chemical intermediates are the same, but some of the key rate constants differ for the actin­bound and free myosin. Steps 1 and 2 are similar to those of free myosin, but step 3—the dissocia­ tion of phosphate—is much faster when a head is bound to an actin filament. As a result, myosin bound to actin traverses the ATPase cycle approximately 200 times faster than myosin free in solution, and ATP hydrolysis becomes the rate­limiting step. This effect of actin is referred to as “actin activation of the myosin ATPase.” A practical advantage of this mechanism is that the ATPase cycle is essentially turned off unless the head interacts with an actin filament. Finally, consider the vertical arrows representing tran­ sitions between bound and free states of each myosin chemical intermediate. All myosin intermediates bind rapidly to actin filaments, but the dissociation rate constants vary over a wide range depending on the

nucleotide that is bound to the active site of the myosin. Myosin with no nucleotide or with bound ADP alone dissociates very slowly and therefore binds tightly to actin filaments. Myosin with bound ATP or ADP + P i dissociates rapidly from actin, so these states bind actin weakly. One cycle of ATP hydrolysis takes about 50 millisec­ onds, but a single pathway cannot be drawn through the reaction mechanism of ATP, myosin, and actin owing to the rapid equilibrium of myosin intermediates (MT and MDP) hopping on and off actin filaments on a millisec­ ond time scale. Starting with AM, ATP binds very rapidly and sets up a rapid, four­way equilibrium including AMT, MT, AMDP, and MDP—the major intermediates during steady­state ATP turnover in muscle (see Chapter 39). Because the products of ATP hydrolysis dissociate much more rapidly from AMDP than from MDP, the favored pathway out of this four­way equilibrium is through AMDP to AMD and back to AM. The overall ATPase rate depends on the actin concentration, which determines the fraction of myosin heads bound to actin in the

628 SECTION IX n Cytoskeleton and Cellular Motility

AMDP state. At the high actin concentrations in cells, a significant fraction of myosin heads is associated with actin (approximately 10% in contracting muscle), but each molecule continues to exchange on and off actin filaments.

Transduction of Chemical Energy Into Molecular Motion

Myosin heads produce force during the transition from the AMDP state to the AMD and AM states. Production of force at this step makes sense for two reasons: First, the large free­energy difference between AMDP and AMD provides sufficient energy to produce force; second, the force­producing AMD and AM intermediates bind tightly to actin, so any force between the motor and the actin track is not dissipated. However, for many myosins, including skeletal muscle myosin, these force­ producing states occupy a small fraction of the whole ATPase cycle. The fraction of the time in force producing states is called the duty cycle. ADP dissociates rapidly from AMD, and ATP binds rapidly to AM, dissociating myosin from the actin filament and initiating another ATPase cycle. Fifty years of research using a combination of mechan­ ical measurements, static atomic structures of myosin heads with various bound nucleotides, and spectro­ scopic observations of contracting muscle revealed the structural basis for the conversion of free energy into force: a dramatic conformational change in the orienta­ tion of the light­chain domain associated with phosphate dissociation (Fig. 36.5B). Elegant mechanical experiments measured the size of the mechanical step produced by a myosin during one cycle of ATP hydrolysis. These experiments on live muscles first suggested that each cycle of ATP hydrolysis moves an actin filament approximately 5 to 10 nm relative to myosin. Now one may observe myosin moving single actin filaments by fluorescence microscopy. An array of myosin heads attached to a microscope slide can use ATP hydrolysis to push actin filaments over the surface (Fig. 36.6A–C). Assays with single myosin molecules show that each cycle of ATP hydrolysis can move an actin filament up to 5 to 15 nm and develop a force of about 3 to 7 piconewtons (pN) (Fig. 36.6D). At low ATP concentrations, the interval between the force­producing step and the binding of the next ATP is relatively long, so single steps can be observed. Further insights emerged from biophysical studies of muscle and purified proteins using x­ray diffraction (see Fig. 39.11), electron microscopy, electron spin reso­ nance spectroscopy, and fluorescence spectroscopy. These experiments showed that the light­chain domain pivots around a fulcrum, the converter subdomain within the catalytic domain, which is stationary relative to the actin filament. For example, spectroscopic probes on

light chains revealed a change in orientation when muscle is activated to contract, whereas probes on the catalytic domain do not rotate. Crystal structures of myosin heads with various bound nucleotides and nucle­ otide analogs show that the light­chain domain can pivot up to 90 degrees (Fig. 36.4D). The light­chain domain is bent more acutely in the AMT and AMDP intermediates and pivots to a more extended orientation when phos­ phate dissociates (Fig. 36.3). ADP dissociation extends this rotation of some classes of myosin. Consistent with rotation of the light­chain domain producing movement, the rate of actin filament gliding in an in vitro assay is proportional to the length of the light­chain domain. The observed range of orientations of the light­chain domain relative to the catalytic domain can account for the observed step size of 10 nm for muscle myosin. Some aspects of these conformational changes and their rela­ tion to phosphate release are similar to Ras family GTPases (see Fig. 4.6). Rotation of the light­chain domain is believed to produce movement indirectly in the sense that force­ producing intermediates stretch elastic elements in the system. This mechanism is represented by a spring in Fig. 36.2. The elastic elements in the myosin­actin complex are most likely to be mainly in the myosin head, with small contributions from the actin and myosin fila­ ments. Movement of the light­chain domain tensions the spring transiently in the AMD and AM states. Dissociation of ADP and rebinding of ATP to the AM intermediate reverts the system to the rapid equilibrium of mostly dissociated weakly bound intermediates. Any force left in the spring is lost as soon as the head dissociates from the actin filament. The actual motion produced depends on the mechani­ cal resistance in the system (Fig. 36.2). If both myosin and actin are fixed, elastic elements are stretched for the life of the force­producing states (AMD and AM), and the energy is lost as heat when the head dissociates. This happens when one tries to lift an immovable object. If the resistance is less than the force in the stretched elastic elements, the actin filament moves relative to myosin, as in muscle contraction. The distance moved in each step depends on the resistance, as the spring stops shortening when the forces are balanced.

Myosin Superfamily

Eukaryotes have 35 classes of myosin and many other examples of unique myosins in single species (Fig. 36.7). All arose from a gene similar to myosin­I in the last eukaryote common ancestor more than a billion years ago. The primordial gene then gave rise to the gene for myosin­V, so this class is also widespread. Gene duplica­ tion, divergence, and acquisition of extra domains pro­ duced many other myosin genes encoding proteins specialized for particular biological functions made pos­ sible by variations of the mechanochemical ATPase cycle

CHAPTER 36 n Motor Proteins

629

A B D Actin filament Step Return Myosin C 40 Actin Events 20 ADP Myosin
A
B
D
Actin
filament
Step
Return
Myosin
C
40
Actin
Events
20
ADP
Myosin
+ Pi
0
ATP
–20
0
0.5
1.0
1.5
GLASS
Time (s)
Distance (nm)

FIGURE 36.6 IN VITRO MOTILITY ASSAYS WITH PURIFIED MUSCLE MYOSIN AND ACTIN FILAMENTS. A–C,  Actin   filament   gliding   assays.   A,   Filaments   are   labeled   with  rhodamine-phalloidin   to   render   them   visible   by   light  microscopy.   ATP   hydrolysis   by   myosin   moves  actin   fila- ments  over  the  surface  with the  pointed  end  leading  as  the myosins walk  toward  the  barbed  end  of  the  filaments.  B–C, Drawings  of  actin  filaments  moving   over   myosin  heads   immobilized   on   a   glass   coverslip.   D,  Measurement  of   the   muscle   myosin   step  size.   An   actin   filament   is   attached   between   two   plastic   beads,   which  are   suspended   by   laser   optical  traps.   The   optical  traps   move  the   filament   near   a   myosin   molecule   on   the   surface   of   another   bead   attached   to   the   microscope   slide,   allowing   a  myosin   head  to   attach   to   the   actin   filament.   When   supplied   with  ATP,   a   single   myosin   head   can  move   the   actin  filament   a   short   distance  corresponding   to   the   step  size.   The   graph   shows   the   time   course   of  displace -

ments   of   the   actin   filament   and   attached   beads.   Brownian   motion   limits   the   precision   of   the   measurement  of   the   size  of   these   steps   to   a   range   of   5   to   15   nm.   The   duration  of   the   step   depends  on   the   ATP  concentration,   because   ATP   dissociates   the   force-producing   AM   state,   allowing   the   force   of   the   optical   traps  to   return   the   beads   and  the  actin   filament   to   their   original  position.   P i ,   inorganic   phosphate.  

( A,  Courtesy   A.   Bresnick,   Albert   Einstein   College   of   Medicine,   New   York.   D,   For   reference,   see  Finer   JT,   Simmons   RM,   Spudich   JA.   Single   myosin molecule   mechanics:  piconewton   forces   and   nanometer   steps.   Nature.  1994;368:113–119.)

and acquisition of diverse tails to interact with cargo. Within a myosin class, the tails are similar to each other, but between classes, tails are diverse in terms of their ability to polymerize and interact with other cellular components including membranes and ribonucleopro­ tein particles. No organism has genes for all 35 classes of myosin and a few species, including Giardia lamblia, have no myosin genes. Myosin­I is most widespread, but plants and related organisms lost this gene. A primitive myosin­V gene gave rise to plant myosin­VIII and myosin­XI, which move at very high speeds (see Fig. 37.9). Organisms on the branch including amoebas, yeast and animals have genes for myosins types I, II, and V, but myosin genes diversified in animals, so humans have 40 myosin genes from 13 classes. Gene duplications gave rise to multiple

isoforms within most classes of myosin. For instance, the vertebrate smooth muscle myosin gene arose from dupli­ cation of a gene for a cytoplasmic myosin­II. Establishing the biological functions of the various myosins has been challenging. Biochemical characteriza­ tion of cargo and localization in cells provide some clues, but genetic or biochemical knockouts often have mild effects, probably owing to overlapping functions of the myosins and the capacity of some cells to adapt to their loss, at least under laboratory conditions. Myosin-I was the first “unconventional myosin” discovered—unconventional in the sense that it differed from the type II myosin originally isolated from skeletal muscle. These myosins have one head and short tails with various types of domains, including a basic domain with affinity for acidic phospholipids. The presence of

630 SECTION IX n Cytoskeleton and Cellular Motility

Class

Example

Heavy chain domains

Architecture

Distribution

Absent

Membrane GPQ Head binding I. Dictyostelium MyoB SH3 Most eukaryotes, Plants, ampicomplexa IQ motif
Membrane
GPQ
Head
binding
I.
Dictyostelium MyoB
SH3
Most eukaryotes,
Plants, ampicomplexa
IQ motif
stramenopiles, others
I.
Bovine BB myosin-I
+ +
Coiled-coil
II.
Chicken muscle myosin-II
Amoebas, fungi, animals
Plants, others
Kinase
III.
Drosophila ninaC long
+ +
Arthropods, chordates
Fungi, plants, others
V.
Chicken myosin V/dilute
Amoebas, fungi, animals
Plants, stramenopiles
VI.
Porcine myosin VI
Animals
Fungi, plants, others
TH4
Talin
VII.
Human myosin VIIA
Animals
Fungi, plants, others
VIII.
Arabidopsis ATM1
Algae, plants
Fungi, plants, others
IX.
Human myosin IXb
Animals
Fungi, plants, others
pH domains
X.
Bovine myosin X
Deuterostomes, Cnidaria Other animals, others
XI.
Arabidopsis MYA1
Algae, plants
Fungi, animals, others
XII.
C. elegans Myo12
Nemotodes only
All others
XIV.
Toxoplasma gondii myosin A
+
= 100 amino acids
Ampicomplexa only
All others

THE MYOSIN FAMILY.  Drawing  of  myosin  heavy  chain   domains   and  molecular   models  of   myosin   isoforms   showing   catalytic 

domains   (rose);  IQ   motifs,   light-chain–binding   sites  (rose bars);   basic   domains   with   affinity  for   membrane  lipids   (violet);  SH3   (Src  homology   3)   domains   (dark green);   coiled-coil  (orange);  kinase   domain   (light blue);   and  pleckstrin  homology  domain   (blue).  (For   reference,   see  Odronitz   F,  

Kollmar   M.   Drawing   the   tree   of   eukaryotic   life   based   on   the   analysis   of   2,269   manually   annotated   myosins   from   328   species.   Genome 2007;8:R196.   See   also   Myosin   Home   Page,   available   at   http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html.)

FIGURE 36.7

Biol.

an Src homology 3 (SH3) domain (see Fig. 25.10) allows some type I myosins to bind proline­rich sequences in other proteins. Those with an actin filament–binding domain separate from the motor domain can crosslink actin filaments. With duty cycles of less than 10%, mul­ tiple myosin heads must work together in concert to move membranes. Mutations show that myosin­I partici­ pates in endocytosis, as expected from its concentration at sites of phagocytosis and macropinocytosis. In micro­ villi of intestinal epithelial cells, myosin­I links actin fila­ ments laterally to the plasma membrane (see Fig. 33.2B). Heavy­chain phosphorylation activates myosin­I from lower eukaryotes, whereas calcium binding to calmodu­ lin light chains regulates myosin­I from the intestinal brush border. The myosin-II class includes various muscle and cytoplasmic myosins that also have two heads, two IQ motifs, and long coiled­coil tails. Assembly of tails into bipolar filaments (see Fig. 5.7) allows myosin­II to pull together oppositely polarized actin filaments during muscle contraction (see Chapter 39) and cytokinesis (see Fig. 44.24). As in smooth muscle (see Fig. 39.23), phos­ phorylation of the regulatory light chain activates myosin­II in animal nonmuscle cells. In addition, phos­ phorylation of the heavy chain regulates the enzyme activity and/or polymerization of some myosin­IIs. Myosin-V moves pigment granules, ribonucleopro­ tein particles and other cellular components (see Fig. 37.11). A long light­chain domain with seven IQ motifs

allows myosin­V to take long steps along the actin fila­ ment (Fig. 36.8). These steps are processive, because slow ADP dissociation from the AMD intermediate allows time for the other head to take a long step and bind an actin subunit 36 nm beyond the first head toward the barbed end of the filament. Mechanical strain after the step may modestly increase the rate of ADP dissociation from the trailing head. This cooperation between the heads initiates ATP binding and the next ATPase cycle, as the motor walks deliberately along the filament. These features make myosin­V a valuable model for the lever arm for movements of the whole myosin family. Myosin-VI arose in metazoan cells and is the only myosin known to move toward the pointed end of actin filaments. Unique features of the converter domain result in the lever arm swinging opposite to the conventional direction. The lever arm is a long, single α­helix begin­ ning with a single IQ­motif associated with calmodulin. Lacking coiled­coil, myosin­VI is a monomer unless adapter proteins bring together the C­terminal globular cargo­binding domains of two molecules. These dimers can take huge steps of approximately 30 nm, but myosin­VI can also act as a tether, because the force­ producing AMD and AM states occupy a large fraction of the ATPase cycle, owing to slow ADP dissociation from AMD state and slow ATP binding to AM. These features allow myosin­VI to move endocytic vesicles from the plasma membrane into the cytoplasm and to contribute to the formation of autophagosomes. Myosin­VII and

CHAPTER 36 n Motor Proteins

631

A Strong Weak Strong A–M A–M*T A–M**DP A–MD A–M M M*T M**DP MD M ≥
A
Strong
Weak
Strong
A–M
A–M*T
A–M**DP
A–MD
A–M
M
M*T
M**DP
MD
M
≥ 1000 s -1
100 s -1
10 s -1
1s -1
0.1 s -1
B Pointed
ADP
ADP
ATP
ADP
ADP
ADP
ADP
ADP
ADP-Pi
ADP
Barbed
Force produced by leading head dissociates ADP from trailing head ATP binds trailing head which
Force produced by
leading head
dissociates ADP
from trailing head
ATP binds trailing
head which
dissociates
Trailing head steps
forward 72 nm and
hydrolyzes ATP
New leading head
binds actin
Pi dissociates

MYOSIN-V MECHANISM.A,  ATPase   cycle  with   ADP  release  as   the   rate-limiting   step  rather   than  phosphate  dissociation   as  

for   muscle   myosin   (see   Fig.   36.5A ).   B,  Relationship   of  mechanical   steps   to  the   ATPase   cycle.   Shown  are   actin  filament   (A),  myosin   head   (M),  ATP (T),   ADP  (D),   and   inorganic   phosphate  (Pi).  (For   reference,   see   De  La   Cruz  EM,   Ostap   EM.   Relating   biochemistry  and   function   in   the   myosin   superfamily.   Curr Opin Cell Biol.  2004;16:61–67.  For  a   movie  of   myosin-V   stepping   on   actin  filaments,   see   Kodera  N,   Yamamoto   D,   Ishikawa   R,  et  al.   V ideo   imaging   of   walking   myosin   V   by   high-speed   atomic   force  microscopy.   Nature.  2010;468:72–76.)

FIGURE 36.8

myosin­X are also monomers with single chain α­helices as lever arms. Myosin mutations cause human diseases. Loss­of­func­ tion mutations in the genes for myosins­IIA, ­IIIA, ­VI, ­VIIA, and ­XV cause deafness and vestibular dysfunction. Mutations in the genes for cardiac muscle myosin heavy and light chains are responsible for many cases of car­ diomyopathies (see Table 39.1).

Microtubule Motors

The kinesin and dynein families of molecular motors use energy from ATP hydrolysis to move vesicles, membrane­ bound organelles, chromosomes, and other cargo along microtubules (see Fig. 37.1). Dynein also powers bend­ ing motions of eukaryotic flagella and cilia (see Fig. 38.14). Dyneins move themselves and any cargo toward the minus end of microtubules. Most kinesins move in the opposite direction, toward the plus end, but some kinesin family members are minus­end­directed motors and others promote microtubule disassembly.

Like myosins, microtubule motors have heads with ATPase activity and tails that interact with cargo.

Kinesins

Kinesin­1 is a processive motor that moves cargo, such as an organelle, continuously toward the plus end of a microtubule. The two heads are attached to an α ­helical coiled­coil tail, much like myosin­II, except both the heads and coiled­coil are smaller (Fig. 36.9). Each head, consisting of approximately 340 residues, is a motor unit that binds microtubules and catalyzes ATP hydrolysis. Light chains associated with the C­terminal bifurcation of the tail bind cargo molecules (Fig. 36.9E). Because the kinesin head is less than half the size of a myosin head and because the proteins lack appreciable sequence homology, the atomic structure of kinesin­1 (Fig. 36.9C) revealed a major surprise: The small kinesin head is folded like the core of the catalytic domain of myosin! In fact, this core, which consists of a central, mixed β­sheet flanked by helices, is similar to the

632 SECTION IX n Cytoskeleton and Cellular Motility

A B D. Structural overlap N E. Kinesin light chain with cargo peptide 1 α3
A
B
D.
Structural overlap
N
E. Kinesin light chain with
cargo peptide
1
α3
β
4
Head
Myosin
β
6
β
7
β
3
Kinesin
α2
β
8
Neck
α1
β
1
Tail
β
2
Cargo
peptide
C.
Kinesin structure
ATP
955
C
Light
chains
Coiled-coil
Tail

FIGURE 36.9STRUCTURE

of   kinesin-1   showing   two   heads   and   the   coiled-coil  tail  with   light  chains   bound  at  the  distal  end.   C,  Ribbon   diagram   of   the  polypeptide   backbone   of   the  kinesin   head  showing  ATP   as  a   space-filling   model  (green),  the   neck-linker   residues   (red),  and   the   proximal  part   of   the   coiled-coil   tail.   D,   Superimposition   of  the   core   of   the   kinesin-1   head   on   the   catalytic   domain  of   myosin  showing   the  structural   homology  of   the  proteins.   The   detailed   ribbon   diagram   shows   only  the   homologous  elements   of   secondary  structure.  The   overview  (right)  shows   kinesin-1   (blue)  superimposed   on   the  structure  of   the   whole  head   of  skeletal   muscle   myosin  (pink).E,  Ribbon   model   of   a   kinesin-1   light  chain   (purple)  with   a  bound   cargo   peptide   (green)  with   a   DWED   motif.   (C,   For   reference,   see   PDB file3KIN   and   Sack   S,   Muller   J,   Marx   A,   et  al.   X-ray   structure   of   motor   and   neck   domains   from   rat  brain  kinesin.   Biochemistry.  1997;36:16155–16165.   D,  Superimposed   ribbon   diagrams   courtesy   of   R.   Vale,   University   of   California,   San  Francisco.   E,  For   reference,   see   PDB file 3ZFW   and   Per nigo   S,   Lamprecht  A,   Steiner  RA,   et  al.   Structural   basis   for  kinesin-1:

cargo   recognition.   Science.  2013;340:356–359.)

OF KINESINS.A,   Domain   architecture  of   the   polypeptide  sequence   of   the  heavy  chain   of   kinesin-1.   B,   Sketch  

considerably smaller Ras family GTPases (see Fig. 4.6). This provided strong evidence that all three families of nucleoside triphosphatases evolved from a common ancestor (Fig. 36.1). ATP binds to a site on kinesin that is homologous to the guanosine triphosphate (GTP)­ binding site of Ras, but the enzyme mechanisms differ in important ways. The microtubule­binding site is some distance from the ATP­binding site (Fig. 36.10).

Kinesin Mechanochemistry

Single kinesin­1 heads, produced experimentally from truncated cDNAs, traverse a microtubule­stimulated ATPase cycle much like myosin (Fig. 36.11A). Both bind and hydrolyze ATP rapidly followed by slower release of phosphate and ADP. However, in contrast to muscle myosin, kinesin heads may remain bound to the micro­ tubule through multiple cycles of ATP hydrolysis rather than dissociating when bound to ATP or ADP­P i . In vitro motility assays (Fig. 36.12) revealed that a two­headed kinesin­1 can move along a single (or two parallel) microtubule protofilaments for long distances at 0.8 µm/s. The motor makes discrete steps of 8 nm, the spacing of successive tubulin dimers in a microtu­ bule. Each step takes 10 milliseconds when kinesin is moving at full speed. This step is remarkably large for the small ( < 10 nm) kinesin heads linked together at the neck region. Kinesin is very powerful, stalling at a force of 6 pN. This allows single molecules to move large organelles through the cytoplasm.

Docked neck linker for kinesin-ATP Disordered neck linker for kinesin without nucleotide
Docked neck linker
for kinesin-ATP
Disordered
neck linker for
kinesin without
nucleotide

(–) α -tubulin

β -tubulin

(+)

INTERACTION OF KINESIN-1 WITH MICRO-

TUBULES.A, Three-dimensional reconstructions  from  electron  micro - graphs   of   kinesin-1   heads   (blue)  bound  to   a  microtubule  (yellow and

red).   The   kinesin   head  on   the  left  has   bound   ATP   and   the   neck  linker  (green) docked. The  kinesin head on  the  right  has  no  bound  nucleotide   and  the   neck   linker   (red)  is   disordered.   Ribbon  diagrams   show   how  the neck  linker  of  the  leading  head (right) must be  unfolded to connect  

to the  trailing  head  at  the  beginning  of  the Sindelar, Yale  University,  based  on  Shang

resolution   structures   of   kinesin   on   microtubules   provide   a   basis   for   nucleotide-gated   force-generation.   Elife.  2014;3:e04686.)

FIGURE 36.10

 coiled-coil  tail.  (From Charles    Z,  Zhou  K,  Xu  C,  et   al.  High-

A

CHAPTER 36 n Motor Proteins

Strong Weak Strong > 100 s -1 80 s -1 300 s -1 Mt–K Mt–KT
Strong
Weak
Strong
> 100 s -1
80 s -1
300 s -1
Mt–K
Mt–KT
Mt–KDP
Mt–KD
Mt–K
KDP
KD
≥ 1000 s -1 100 s -1 10 s -1 1s -1 0.1 s -1
≥ 1000 s -1
100 s -1
10 s -1
1s -1
0.1 s -1

633

(+) B ADP ADP 0 α ATP 0 β ATP ADP + Pi Pi ADP
(+)
B
ADP
ADP
0
α
ATP
0
β
ATP
ADP
+ Pi
Pi
ADP
ADP
ADP
(–)
Trailing head weakly associates with MT ATP binds leading head Trailing head Pi dissociates from
Trailing head weakly
associates with MT
ATP binds
leading head
Trailing head
Pi dissociates
from trailing head
weakening head's
binding to MT
rotates
New trailing head
hydrolyzes ATP
New leading head
binds MT and
dissociates ADP

FIGURE 36.11 KINESIN-1 ATPase MECHANISM. A,  A  diagram  of  the  kinesin-microtubule ATPase cycle for  a  single  kinesin-1  head  showing  the   kinesin   (K),   microtubule   (Mt),   ATP   (T),  ADP   (D),   and   inorganic   phosphate   (P).   Arrow sizes   are   proportional  to  the  rates   of   the  reactions,   with  second-order   reactions  adjusted   for  physiological   concentrations  of   reactants.   The   beige  shading  shows  two   pathways   through   the  reaction,   one   along   the   top  line  without   dissociation,   and   the   other  with   dissociation   from  the   microtubule.   B,   Hand-over-hand,   processive   stepping   of   kinesin   along   a   microtubule.   The   empty,   microtubule-bound   head   binds   and  hydrolyzes   ATP   resulting  in   a   conformational  change  favoring   docking   of  its  neck-linker   (green),  thereby   moving  forward   the  detached  head  with   its   undocked   (pink)  neck-linker.  Binding  of   the  new   leading   head   to   the   microtubule   causes   a   conformation   change   that   dissociates   ADP.   Dissociation   of   the   γ -phosphate  from   the   trailing   head   results   in   its   dissociation  from   the   microtubule.   This  returns  the   heads   to   their   original   condition,   but  with   the  motor   advanced   8   nm   with   the  heads   in   the   opposite   chemi - cal  states.   P i ,   inorganic   phosphate.   (B,  Data   from   R.   Vale,   University   of   Califor nia,   San   Francisco,   and   R.   Milligan,  Scripps   Research   Institute,   La   Jolla,   CA.   For   reference,   see   Cao   L,  Wang   W,   Jiang   Q,   et   al.   The   structure   of   apo-kinesin   bound   to   tubulin   links   the  nucleotide  cycle   to   move - ment.   Nat Commun.  2014;5:5364.)

Processive movement depends on the ability of kinesin to remain associated with the microtubule through more than a hundred cycles of ATP hydrolysis. This is made possible by cooperation between the two heads that ensures at least one head is bound to the microtubule throughout the ATPase cycle (Fig. 36.11B). Reciprocal affinities for nucleotide and microtubules allow the two heads to alternate between microtubule binding and dissociation. For example, if kinesin­1 with ADP bound to both heads is mixed with microtubules, one head binds a microtubule and rapidly dissociates its ADP, leaving the other head dissociated with bound ADP. Binding and hydrolysis of ATP on the open site of the head associated with the microtubule, drives a con­ formational change that repositions the trailing head forward so it can pass the bound head and bind the next tubulin dimer toward the plus end of the microtubule. Association of the new leading head with the microtu­ bule promotes dissociation of its bound ADP. Dissocia­ tion of the γ­phosphate from the new trailing head

weakens its affinity for the microtubule. Its detachment from the microtubule with bound ADP brings the cycle back to its starting point with kinesin having advanced 8 nm (Fig. 36.12). Experiments with single kinesin­1 molecules labeled with fluorescent dyes showed that they alternate steps on the right and left sides of the microtubule like a gymnast walking on a balance beam. The mechanism of stepping is postulated to be the docking and undocking of a segment of the kinesin­1 heavy chain linking the motor domain to the coiled­coil neck and tail (Fig. 36.10). With ATP bound the motor domain has a conformation that favors association of the “neck­linker” peptide, as in the X­ray structure of dimeric kinesin (Fig. 36.9C). When either no nucleotide or ADP is bound, the conformation of the motor domain releases the neck­linker peptide.

Kinesin Superfamily

The last eukaryotic common ancestor had only a single myosin but already had at least 11 families of kinesins

634 SECTION IX n Cytoskeleton and Cellular Motility

A B C Microtubule movement 96 80 Bead movement 64 (–) (+) Kinesin stepping toward
A
B
C
Microtubule movement
96
80
Bead
movement
64
(–)
(+)
Kinesin stepping
toward (+) end
48
32
(–)
16
Kinesin stepping
toward (+) end
0
(+)
1
2
3
4
5
Time (s)
Distance (nm)

FIGURE 36.12

microscope   slide  uses   ATP   hydrolysis  to   move   microtubules   over   the   surface.   A   single   kinesin-1   molecule   can   move   a   microtubule   in   this   assay.  

Microtubules   can   be   imaged   by   video-enhanced   differential  interference   contrast   microscopy  (see  Fig.   34.6)   or   fluorescence   microscopy.   B,   Bead assay. Kinesin  or  dynein that  is attached  to  a  plastic  bead  uses  ATP hydrolysis to  move the  bead  along a  microtubule  attached  to the microscopic  

slide.  C,  Experimental   measurement  of  the   kinesin-1   step   size  using   the   bead  assay.  The   bead   is   held  in   a  laser   optical  trap   so   that  8-nm  steps  can  be  recorded, as  a  single,  two-headed  kinesin-1  moves a bead processively  along  a  microtubule,  as  in  B. The  position of the bead  is  recorded  

with  nanometer  precision by  interferometry.  (For  reference,  see  Svoboda K,  Schmidt  CF,  Schnapp  BJ,  et   al.  Direct  observation  of  kinesin  stepping by   optical  trapping   interferometry.   Nature.  1993;365:721–727.)

IN VITRO MOTILITY ASSAYS FOR MICROTUBULE MOTORS.A,   Gliding   assay.   Kinesin   or   dynein   that  is   attached   to   a  

Kinesin structures

N-terminal motor

Kinesin-1

Kinesin-2

Example

Hs Kif5B

Sp Krp85/95

Heavy chain domains

Kinesin-2 Example Hs Kif5B Sp Krp85/95 Heavy chain domains Head Coiled-coil Tail Architecture 85 95 Kinesin-3
Head Coiled-coil Tail
Head
Coiled-coil Tail

Architecture

Heavy chain domains Head Coiled-coil Tail Architecture 85 95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1
Heavy chain domains Head Coiled-coil Tail Architecture 85 95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1

85

95

Kinesin-3

Kinesin-4

Kinesin-5

Mm

Kif1b

Xl KIp1

Dm

Klp61f

95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1 Dm Klp61f Kinesin-7 Internal motor Kinesin-13 C-terminal motor
95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1 Dm Klp61f Kinesin-7 Internal motor Kinesin-13 C-terminal motor
95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1 Dm Klp61f Kinesin-7 Internal motor Kinesin-13 C-terminal motor
95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1 Dm Klp61f Kinesin-7 Internal motor Kinesin-13 C-terminal motor
95 Kinesin-3 Kinesin-4 Kinesin-5 Mm Kif1b Xl KIp1 Dm Klp61f Kinesin-7 Internal motor Kinesin-13 C-terminal motor

Kinesin-7

Internal motor

Kinesin-13

C-terminal motor

Kinesin-14

Hs CENP-E Xl MCAK Dm Ncd
Hs CENP-E
Xl MCAK
Dm Ncd

FIGURE 36.13 KINESIN FAMILY. A,   Phylogenetic   relationships   of   some   of   the  kinesins   based   on   the  sequences   of   the   motor  domains.  

coiled-coil   tail   (orange),

and   tail  piece   (blue).  (Data   from   R.  Case   and   R.  Vale,   University   of   Califor nia,  San   Francisco.   For   reference,   see  Lawrence   CJ,   Dawe  RK,   Christie   KR,  et   al.   Standardized   kinesin   nomenclature.   J Cell Biol.   2004;167:19–22;   and   Dagenbach   EM,   Endow  SA.   A   new   kinesin   tree.  J Cell Sci.   2004;117:3–7.   See   also   the   Kinesin   Home   Page   at   https://labs.cellbio.duke.edu/kinesin .)

B,   Drawing   of   kinesin   heavy   chain  domains  and   molecular   models   of   kinesin  isoforms  showing   the   catalytic  domain   (red),

with motor domains associated with a variety of coiled­ coil stalks and tails (Fig. 36.13 and Table 36.2). Thus the microtubule system was much more developed than the actin system at this point in evolution. Contempo­ rary eukaryotes have genes for 17 families of kinesins

(often with multiple isoforms) plus a few more para­ logs identified in isolated species. On the other hand, many eukaryotes have lost one or more kinesin genes; for example, amoebas lack kinesin­2 and alveolates lack kinesin­7.

CHAPTER 36 n Motor Proteins

635

TABLE 36.2

Kinesin Superfamily: Classification and Examples of Kinesin-Family Motor Proteins

Class

Examples

Subunits (kD)

Velocity ( µ m s 1 )

Functions

N-Terminal Motor

 

Kinesin­1

Human KHC

2 × 110, 2 × 70

+ 0.9

Organelle movement

Kinesin­2

Urchin KRP85/95

1 × 79, 1 × 84, 1 × 115

+ 0.4

Organelle movement

Kinesin­3

Mouse KIF1B

1 × 130

+ 0.7

Mitochondria movement

Kinesin­4

Xenopus Kp11

2 × 139

+ 0.2

Chromosome movement

Kinesin­5

Fly KLP61F

4 × 121

+ 0.04

Pole separation, mitosis

Kinesin­7

Human CENP­E

2 × 340

+ 0.1

Kinetochore­microtubule binding

Internal Motor

Kinesin­13

MCAK

2 × 83

Microtubule disassembly

C-Terminal Motor

Kinesin­14

Fly Ncd

2 × 78

0.2

Mitotic/meiotic spindle

Modified from Vale RD, Fletterick RJ. The design plan of kinesin motors. Annu Rev Cell Dev Biol. 1997;13:745–777. More data on kinesins are available at the Kinesin Home Page, https://labs.cellbio.duke.edu/kinesin.

All members of the kinesin family have similar motor domains attached to a variety of tails that interact with cargo (Fig. 36.13). Motor domains are generally found at the N­terminus, but may be located in the middle (kinesin­13) or at the C­terminus (kinesin­14). Regardless of their location, motor domains have similar structures. Most kinesins are dimeric, with two polypeptides joined in a coiled­coil. Most are homodimers, but kinesin­2 not only forms homodimers but also heterotri­ mers consisting of two different polypeptides with motor domains plus another large subunit. Tetrameric kinesin­V molecules bind to two microtubules with opposite polarities and move them apart. Most kinesins move toward the plus end of the micro­ tubule, but C­terminal kinesin­14 motors move toward the minus end. The reverse direction of kinesin­14 move­ ment is not explained by either the architecture of the motor domain, the ATPase mechanism, which is similar to kinesin­1, or the attachment of the N­terminus of the motor domain to the stalk. Instead, the proximal part of the Ncd coiled­coil stalk rotates approximately 70 degrees toward the minus end of the microtubule when ATP binds to the active site. Kinesins transport a variety of cargo, including chro­ mosomes and organelles, along microtubules. Kinesin­1 moves membrane vesicles toward the plus end of micro­ tubules away from the centrosome and along axons of neurons (see Fig. 37.1). The light chain links the end of the kinesin­1 tail to cargo proteins (Fig. 36.9E). Kinesin­2 is the motor for anterograde intraflagellar transport, movement of particles toward the tip of the axoneme in cilia and flagella (see Fig. 38.18). It also transports a variety of cargos over long distances in the cytoplasm. Kinesin­4 motors (also called chromo- kinesins) bind both DNA and microtubules. In neurons, they move cargo along axons. In dividing cells they participate in the formation of condensed mitotic

chromosomes (see Fig. 44.7). Kinesin­7 (originally called CENP-E) concentrates at kinetochores where it helps move the chromosome toward the middle of the mitotic spindle prometaphase (see Fig. 44.5). Bipolar kinesin-5 motors form an antiparallel tetramer of two dimeric kine­ sins that bridge a pair of oppositely polarized microtu­ bules and push apart the poles of the mitotic spindle (see Fig. 44.7). Both kinesin­8 and kinesin­13 use cycles of ATP hydro­ lysis to remove tubulin dimers from the ends of micro­ tubules. Kinesin­8 motors to the plus end where it works, whereas kinesin­14 lacks motor activity but can diffuse on the microtubule surface to reach either end. This depolymerizing activity is important for mitosis. Most kinesins appear to be constitutively active, but intramolecular interactions autoinhibit kinesin­1. The tail folds back and binds between the heads, shutting off the motor. Adapter proteins compete the tail from the head, freeing the motor to be active.

Dyneins

Dynein microtubule­based motors are AAA ATPases (Box 36.1), so their evolutionary origin differs from myosins and kinesins (Fig. 36.1). Most AAA ATPases consist of six separate ATPase domains, but in dynein these domains (AAA1–AAA6) are concatenated in a giant heavy chain of nearly 500 kD rather than separate polypeptides (Fig. 36.14A). Cytoplasmic dynein is a dimer of two heavy chains plus accessory polypeptides that bring the total molecular weight to approximately 1.4 MDa. The N­terminal quarter of the heavy chain forms a tail that interacts with intermediate chains, light intermediate chains, dimers of light chains, and cargo molecules (Fig. 36.14B). A linker domain connects the tail to AAA1. A segment of the dynein heavy chain within AAA4 forms an antiparallel coiled­coil stalk with a small microtubule­ binding domain at the tip.

636 SECTION IX n Cytoskeleton and Cellular Motility

A N 0 B. Whole dynein molecule C D Linker AAA1 “Pre power stroke” ATP
A
N
0
B. Whole dynein molecule
C
D
Linker
AAA1
“Pre power stroke”
ATP analog
in AAA1
“Post power stroke”
ADP in AAA1
DHC
dimerization
Tail
domain
Tail
DIC WD40
AAA2
DLIC
AAA6
1388
AAA5
Linker
AAA3
1
LC8
Buttress
AAA4
of AAA5
Tctex
2
Motors
3
Coiled-coil
4
stalk
Coiled-coil
stalk of AAA4
Microtubule
binding site
5
6
Microtubule binding
domain of AAA4
C
4730
AAA-ATPase domains

FIGURE 36.14 DYNEIN STRUCTURE. A,   Domain   organization   of   a  dynein   heavy   chain   showing   the  six   AAA   ATPase  modules  and  two  sequences   that   form   an   antiparallel   coiled-coil   stalk   with  an   ATP-sensitive  microtubule-binding   site  at   the  tip.   The   first  AAA   domain  is  the   catalytic  site.   AAA   domains   2,   3,   and   4   bind   ATP,  but   hydrolysis  is   not   coupled   to   movement.   AAA   domains   5  and   6   do  not  bind  ATP.   B,   Model   for  cyto- plasmic   dynein-1   based   on   crystal   structures   of   motor   domains   and  reconstructions  of   electron   micrographs   of   the  dynactin   complex.  C,   Ribbon   diagram   of   crystal  structures   of   cytoplasmic   dynein-1   motor   domains   (Apo)  without  nucleotide  bound  to   AAA1.   The   linker   domain   is  purple.   AAA  domains   are  color   coded   as   in   A,   with   the   stalk   and   microtubule  binding   domain  extending   from   AAA3.   D,   Space-filling   models   of   dynein  with  (ADP-V o )   with  ADP   and   the   phosphate   analog   vanadate   bound   to   AAA1   the   “pre  power   stroke”   state   and   ADP   bound   to   AAA1   the  “post   power

  stroke  state.”   The  AAA   hexamer  is  more   compact   with   bound  ADP-V o .   These   two   structures   differ   in   the   conformations   of   the   AAA   hexamer,   linker   domain   and   stalk.   (For   reference,   see   EMData Bank files 2861   and  2862   and   Schmidt  H,   Zalyte   R,   Ur navicius   L,   et   al.   Structure   of  human   cytoplasmic   dynein-2   primed   for   its   power  stroke.   Nature.  2015;518:435–438;   and   Ur navicius   L,   Zhang   K,   Diamant   AG,   et  al.   The   structure   of   the   dynactin  complex   and   its  interaction  with  dynein.   Science.  2015;347:1441–1446.)

Dynein Mechanochemistry

Sufficient information is available from enzyme kinetics, crystal structures, and motility assays to construct a mechanochemical cycle for dynein interacting with a microtubule (Fig. 36.15A). The AAA1 domain binds and hydrolyzes ATP during force­producing interactions with microtubules. Full motor function requires ADP or ATP binding to AAA domains 2 to 4, but ATP hydrolysis by these domains is not coupled directly to motility. AAA domains 5 and 6 do not bind nucleotides. The dynein ATPase cycle of AAA1 resembles the actomyosin ATPase mechanism in broad outline. When AAA1 is free of nucleotide, dynein binds tightly to a microtubule at a site between the α­ and β­tubulin subunits with the stalk pointing toward the minus end of the polymer. ATP binding to AAA1 causes compaction of the whole AAA hexamer and produces two important conformational changes (Fig. 36.14). First, the “buttress” on AAA5 communicates the conformational change in the hexamer to the stalk by displacing the stalk helices relative to each other. This, in turn, changes the confor­ mation of the microtubule­binding domain more than 20 nm distant from the ATP binding site and reduces its affinity for the microtubule. Thus, dynein­ATP dissoci­ ates from the microtubule. Second, the linker domain bends in the middle, moving to the “pre power stroke state.” After ATP hydrolysis, dynein­ADP­P i also binds

weakly to microtubules, but phosphate dissociation during one of the transient interactions with a microtu­ bule reverses both conformational changes produced by ATP. The linker domain straightens out, producing a power stroke that moves the tail and any associated cargo (including the other subunit of a dynein dimer) toward the minus end of the microtubule. In addition, the affinity for microtubules increases, allowing trans­ mission of force from the microtubule to the cargo. Binding to a microtubule also stimulates the rate of ADP dissociation from dynein approximately 10­fold, from approximately 3 s 1 to approximately 33 s 1 , restarting the ATPase cycle. Yeast dynein dimers can walk processively toward the minus end of a microtubule in in vitro motility assays. The mechanism likely involves steps by the two motor domains on adjacent protofilaments of the microtubule. The size of the mechanical step associated with each ATP hydrolysis in most often 8 nm, but cytoplasmic dynein can take larger steps up to 24 nm when the load is low. Yeast dynein produces a force of 5 to 7 pN, similar to kinesin.

Dynein Superfamily

Dynein genes are ancient, arising well before the last common eukaryotic ancestor (see Fig. 2.4B), but they were lost multiple times during evolution, so neither red

CHAPTER 36 n Motor Proteins

637

Apo ATP ADP•P ADP Apo S W W S S Strong Weak Strong P 30
Apo
ATP
ADP•P
ADP
Apo
S W
W
S
S
Strong
Weak
Strong
P
30 s -1
Mt–Dy
Mt–DyT
?
Mt–DyDP
Mt–DyD
Mt–Dy
P
3s -1
Dy
DyT
DyDP
DyD
Dy
ATP
ADP•P
≥ 1000 s -1
100 s -1
10 s -1
1s -1
0.1 s -1

DYNEIN-MICROTUBULE ATPase MECHANISM.  Chemical   pathway   and   structures.   Arrow sizes  are   proportional   to   the  

rates   of   the   reactions,   with  second-order  reactions  adjusted   for   physiological   concentrations   of   reactants.   The   beige  shading   shows   the   main   pathway  through   the   reaction.   D,   ADP;  Dy,   dynein;   Mt,  microtubule;   P,  inorganic   phosphate;   T,   ATP.   The   drawings   are   interpretations  of   the   intermediates  in   the   cycle   based   on  crystal   structures.   (Modified   from  Cianfrocco   MA,  DeSantis   ME,   Leschziner   AE,   et   al.   Mechanism   and   regula - tion   of   cytoplasmic   dynein.   Annu Rev Cell Dev Biol.  2015;31:83–108.)

FIGURE 36.15

algae nor flowering plants now have dynein genes. Animals have two genes for cytoplasmic dynein heavy chains and multiple isoforms of intermediate, light inter­ mediate, and light chains. Alternative splicing, especially of intermediate chains, further increases the complexity. Cytoplasmic dyneins are dimeric proteins (Fig. 36.14B). Cytoplasmic dynein­2 moves cargo for intrafla­ gellar transport (see Fig. 38.18). Cytoplasmic dynein­1 has diverse functions around the entire cell cycle. During interphase, dynein­1 transports organelles, RNAs, and some viruses toward the minus ends of microtubules (see Fig. 37.2) generally moving cargo toward the cell center where the centrosome and Golgi apparatus are located. In neurons, dynein­1 moves cargo inside axons toward the cell body (see Fig. 37.3). During mitosis, dynein­1 in the cell cortex and bound to kinetochores of chromosomes applies forces to microtubules and helps to position the mitotic spindle (see Fig. 44.7). Given these diverse activities, it is not surprising that dynein mutations cause serious phenotypes and contrib­ ute to disease. A null mutation in the gene for a mouse cytoplasmic dynein­1 heavy chain leaves the Golgi appa­ ratus dispersed throughout the cytoplasm and is lethal during embryogenesis. A temperature­sensitive mutation in Caenorhabditis elegans dynein­1 causes defects in mitosis at the restrictive temperature. Mutations of dynein­1 and associated proteins contribute to human neurodegenerative diseases including some cases of Par­ kinson disease and spinal muscular atrophy. Accessory proteins regulate the enzyme activity and movements of cytoplasmic dyneins. Transport by mammalian dynein depends on the dynactin complex

(see Fig. 37.2), a huge complex of 23 subunits (11 dif­ ferent proteins) with a total molecular weight of approx­ imately 1.2 MDa. Dynactin and an adapter protein not only link cytoplasmic dynein­1 to cargo, but also stimu­ late its motor activity, perhaps by positioning the two motors in a productive way. A complex of proteins (Lis-1 and Nudel/NudE) increases dynein’s affinity for microtubules, slows the motor, and may increase force production. Nudel/NudE binds intermediate chains and tethers Lis­1 to dynein. Lis­1, a β ­propeller protein, interacts directly with the AAA hexamer and sterically blocks a movement of the linker domain that is required for microtubule release. Genetic experiments suggest that Lis1 is a dynein acti­ vator, but how tight binding to microtubules activates dynein remains unclear. Loss­of­function mutations of the Lis1 genes interfere with development of the cerebral cortex, which lacks gyres and is smooth in affected humans. Humans have 14 genes for axonemal dyneins, which consist of one to three heavy chains. In axonemes of cilia and flagella, at least seven different dynein isoforms bind to unique sites on the outer doublets (see Fig. 38.14). Calcium and a cyclic adenosine monophosphate (cAMP)– dependent protein kinase (see Fig. 25.3D) regulate dynein in cilia and flagella.

ACKNOWLEDGMENTS

We thank Andrew Carter, Erika Holzbaur, Samara Reck­ Peterson, and Lee Sweeney for their suggestions on revi­ sions to this chapter.

638 SECTION IX n Cytoskeleton and Cellular Motility

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