Relating biochemistry and function in the myosin superfamily

Relating biochemistry and function in the myosin superfamily$

Enrique M De La Cruz1 and E Michael Ostap2

All characterized myosins share a common ATPase mechanism.

However, detailed kinetic analyses suggest that modulation

of the rate and equilibrium constants that define the ATPase

cycle confers specific properties to these motor proteins, suiting

them to specific physiological tasks. Understanding the kinetic

mechanisms allows potential cellular functions of the

different myosin classes and isoforms to be better defined.

Addresses1Department of Molecular Biophysics and Biochemistry, Yale University,PO Box 208114, New Haven, CT 06520, USA

e-mail: [email protected] Muscle Institute and Department of Physiology, University

of Pennsylvania School of Medicine, B400 Richards, Philadelphia,

PA 19104, USA

e-mail: [email protected]

Current Opinion in Cell Biology 2004, 16:61–67

This review comes from a themed issue on

Cell structure and dynamicsEdited by John A Cooper and Margaret A Titus

0955-0674/$ – see front matter

� 2003 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.ceb.2003.11.011

AbbreviationsA actin

M myosin

Pi inorganic phosphate

IntroductionThe myosin superfamily of actin-based molecular motors

consists of at least 18 members with diverse properties

adapted for a variety of cellular activities [1,2]. For

example, some myosins exist in large macromolecular

assemblies that power contraction of muscle, while others

work in small numbers to support the translocation of

single organelles. Recent studies from several laboratories

have probed the mechanisms of a diverse set of myosin

classes and isoforms. Our goal is to provide a brief over-

view of the ATPase mechanisms of a few members of the

myosin superfamily and to point out how differences in

biochemical rate constants provide myosins with motor

properties tuned for diverse biological functions.

Overview of the myosin ATPase cycleThe actomyosin ATPase cycle mechanism appears to be

conserved for all myosin isoforms. The reaction scheme in

Figure 1 shows the mechanism of the actomyosin ATPase

pathway coupled to a mechanical model [3,4]. Although it

is an oversimplification of what is known about chemo-

mechanical coupling, the scheme provides the basic

details required for understanding the biochemical diver-

sity of myosins and will be referred to throughout the

kinetic discussions in this review. For more detailed

discussion of chemomechanical coupling and detailed

references concerning the actomyosin ATPase cycle,

we recommend the excellent textbook by Howard [5]

and the review by Geeves and Holmes [6].

The minimum mechanism needed to describe the myo-

sin ATPase cycle consists of at least six nucleotide-linked

biochemical reactions. These are described in detail in

Figure 1.

Intermediates are defined according to their affinities

for actin. The M.ATP and M.ADP.Pi states are ‘weak-

binding’ or ‘pre-force generating’ intermediates that

attach and detach (steps 2 and 4) from the actin filament

on a submicrosecond scale, have low (micromolar) actin

affinities, and do not bind stereospecifically to the actin

filament [6]. The weak-binding states do not bear force,

but allow myosin to ‘cock’ itself for the power-stroke

while detached from actin (step 3), preventing a reverse

power-stroke while attached to actin. The AM and

AM.ADP states are ‘strong-binding’ intermediates that

stereospecifically attach to actin filaments with high

(submicromolar) affinities. The strong-binding states

are the ‘force-bearing’ intermediates.

The force-generating power-stroke coincides with phos-

phate release at the transition from the weakly bound to

strongly bound states (step 5, Figure 1). Because it con-

trols the weak-to-strong transition, Pi release is an ideal

step for controlling myosin–actin attachment and moti-

lity. The transition is regulated by the calcium regulatory

system in striated muscle and by heavy-chain phosphor-

ylation of some myosin-I isoforms [7].

Enzymatic adaptations of myosin classesand isoformsTwo important parameters that influence the mechanical

and motile properties of myosins are, first, the lifetimes

(i.e. the duration of the biochemical states in units of

time) of the strongly bound intermediates (AM and

AMD), and, second, the duty ratio. The attachment

lifetimes and duty ratios can vary considerably between

myosin classes and between isoforms within a given class.

The duty ratio of a molecular motor is defined as the

fraction of the total ATPase cycle time that an individual

$ Supplementary data associated with this article can be found, in the

online version, at doi:10.1016/j.ceb.2003.11.011

www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:61–67

motor is attached to its track [5]. For a population of

motors, the duty ratio is the steady-state fraction of

attached molecules. The attached motors can be in the

pre- [8–11] or post-power-stroke states. When discussing

myosins, the duty ratio usually refers to the fraction of

time spent in the strongly bound states. For example, low-

duty-ratio myosins spend a large proportion of time in the

M.ATP and M.ADP.Pi states, whereas high-duty-ratio

myosins occupy the AM and AMD states. In simple kinetic

terms, one would expect steps 4 or 5 (Figure 1) to be rate

limiting (i.e. the slowest step or ‘bottleneck’ in the ATPase

cycle) for low-duty-ratio myosins, and steps 1 or 6

(Figure 1) to be rate limiting for high-duty-ratio myosins

(see animation in supplementary material). The duty ratio

of characterized myosins depends on the rates into and out

of the strong-binding states, and therefore depends on the

actin and nucleotide concentrations [12,13�].

Myosin-I

Myosin-I isoforms are the single-headed, membrane-asso-

ciated members of the myosin superfamily found in most

eukaryotic cells. They play essential roles in membrane

dynamics, cytoskeletal structure, mechanical signal-trans-

duction and endosome processing [1]. The myosin-I

family can be divided into two major subclasses on the

basis of motor domain sequences (Figure 2a). Subclass 1

contains the ‘amoeboid’ or long-tailed myosin-I proteins.

Subclass 2 contains the ‘short-tailed’ myosins, including

the well-characterized myo1a (brush border myosin-I) and

the more widely expressed myo1b and myo1c.

All characterized myosin-I isoforms are low-duty-ratio

motors (i.e. they spend a small fraction of their catalytic

cycle strongly bound to actin), and the weak-binding

states have a very low actin affinity [14,15,16�]. There-

fore, small numbers of myosin-I motors cannot support

contraction or movements of cargo over long distances.

Comparative kinetic studies of myosin-I isoforms have

identified important differences between subclasses. The

rate constants of most characterized subclass-2 isoforms

are slower than subclass-1 isoforms [14,15,16�,17,18],

resulting in all biochemical intermediates having signifi-

cantly longer lifetimes.

By examining the lifetimes of the strong-binding states

(Figure 2b), we gain important insights into the physio-

logical function and regulation of myosin-I proteins. The

long lifetimes of the force-bearing intermediates of sub-

class-2 myosins are likely to be crucial for structural or

tension-maintenance roles [15,17]. For example, the

force-generating properties of these motors may function

Figure 1

AMAM.ADPAM.ATP

M.ATP

AM.ADP.Pi

M.ADP.Pi

A = Actin

M = Myosin

Weak-binding states

Strong-binding states

AM

(5)Powerstroke

ADPPi

Rate-limiting step for low-duty-ratio myosins

Rate-limiting step for high-duty-ratio myosins

ATP

(1)

(2)

(3)

(4)

(6)

Current Opinion in Cell Biology

The actomyosin ATPase cycle, identifying the weak- and strong-binding states of myosin. In the absence of ATP, myosin binds tightly to actin.

ATP binding (step 1) induces a conformational change in myosin that weakens its actin affinity and causes myosin to detach from actin (step 2).

A second conformational change allows hydrolysis of ATP to ADP and inorganic phosphate (Pi) (step 3); the hydrolysis products remain bound to

myosin. The M.ADP.Pi state rebinds to the actin filament (step 4) and a force-generating power-stroke accompanies phosphate release (step 5).

ADP is released (step 6) and the cycle repeats upon ATP binding. The actin-detached M and M.ADP states are omitted for clarity and to illustrate

that these states are not populated during enzymatic cycling in the presence of saturating actin and ATP.

62 Cell structure and dynamics

Current Opinion in Cell Biology 2004, 16:61–67 www.sciencedirect.com

in putting tension-sensitive calcium channels under ten-

sion [19��] and in defining the structure and organization

of cellular organelles [20]. The short lifetimes of the

strong-binding states of subclass-1 isoforms may favor

their playing a role in rapid contractile events where large

numbers of myosins are required, as in the rapid contrac-

tion of actin-rich membrane protrusions [21,22].

Myosin-II

Myosin-II isoforms are the major contractile proteins in

muscle and also play several crucial roles in non-muscle

contractility. Myosin-II molecules contain two motor

domains and assemble into bipolar filaments. Like the

myosin-I family, phylogenetic analysis of the motor

domains of the myosin-II family shows a division of

isoforms into distinct subclasses that cross phylogenetic

boundaries. The myosin-II family can be divided into

non-muscle cytoplasmic, cardiac muscle, smooth muscle,

and skeletal muscle subclasses, each with multiple iso-

forms [2,23].

All characterized muscle myosin-II isoforms have com-

parable unitary forces and displacements — a single

skeletal muscle myosin-II molecule generates the same

force and displacement as a single smooth muscle myo-

sin-II molecule [24]. However, the kinetic intermediate

lifetimes and duty ratios of muscle isoforms show a great

deal of variation that results in important mechanical

differences, accounting for their functional diversity.

For example, the duty ratio and the lifetime of the

strong-binding states of smooth muscle myosin are sig-

nificantly longer than those of skeletal muscle isoforms

[24,25]. In muscle tissue, these kinetic differences are

likely to be responsible for smooth muscle having slower

rates of contraction and producing higher forces than

skeletal muscle [24].

Recent investigations of non-muscle myosin-IIb demon-

strate that it is a relatively high-duty-ratio motor under

physiological actin and nucleotide concentrations (myosin

is attached to actin for �20–50% of its ATPase cycle time)

Figure 2

0

100

200

300

400

500

Subclass-1Myosin-I(Myo1e)

Subclass-2Myosin-I(Myo1b)

Myosin-V

Life

time

of s

tron

g-bi

ndin

g st

ates

(m

s)

Human Myo1aMouse Myo1aRat Myo1aChick Myo1aMouse Myo1bRat Myo1bHuman Myo1cMouse Myo1cDicty MyoEAcan Myo1aHuman Myo1eRat Myo1eAcan Myo1bDicty Myo1bAcan Myo1c

Sub

clas

s-2

Sub

clas

s-1

Subclass-1Myosin-I(Myo1e)

Subclass-2Myosin-I(Myo1b)

Myosin-V

Fraction of ATPasecycle spent inweak–bindingstates

Fraction of ATPasecycle spent instrong–bindingstates

Myosin-VI

(b)

(c)

(a)


Relationship between myosin duty ratio and lifetime of the strong-binding state. (a) Phylogenetic analysis of several members of the myosin-I

family showing subclass divisions. (b) Lifetimes of the AM.ADP strong-binding states of myosins -I, -V, and -VI [15,16�,37,43]. Notice that the lifetime

of the strong-binding state of myo1b (a low-duty-ratio motor) is significantly longer than that of processive myosin-V. (c) Pie charts showing the

relative amounts of time per ATPase cycle that myosin-I proteins (low-duty-ratio motors) and myosin-V (a high-duty-ratio motor) spend in the

weak- and strong-binding states in the presence of 50 mM actin.

Relating biochemistry and function in the myosin superfamily De La Cruz and Ostap 63


[13�,26�]. In this way it differs significantly from non-

muscle myosin-IIa [27] and muscle myosin isoforms,

which possess low duty ratios (myosin is attached to actin

for �10% of its ATPase cycle time). Thus, non-muscle

myosin-IIb appears to be adapted for cellular roles requir-

ing tension maintenance, whereas non-muscle myosin-IIa

is better suited to more rapid sliding. Kovacs et al. [27]

suggest that cellular myosin filaments may be composed

of multiple isoforms, which raises the possibility of

further kinetic ‘fine-tuning’ by the regulation of myosin

filament content.

When operating under high forces (as in a muscle under-

going an isometric contraction), low-duty-ratio myosin-II

isoforms may become high duty-ratio motors; in other

words, high forces may slow the rate at which intermedi-

ates leave the strong binding states [5,6]. However, the

effect of force on the kinetic and molecular mechanical

cycles is not understood and is the focus of intensive

study.

Myosin-V

Myosin-V transports vesicles along actin filament tracks

over long distances as a single, two-headed molecule [28–

31,32��,33��,34�,35�,36]. Detailed kinetic analysis of sin-

gle-headed myosin-V [37–40] demonstrate that ATP

binding (step 1, Figure 1), dissociation from actin (step

2, Figure 1), hydrolysis off actin (step 3, Figure 1) and Pi

release on actin (steps 4–5, Figure 1) are rapid (�200-

800 s�1) and essentially irreversible, whereas ADP release

(step 6, Figure 1) is slow (�16 s�1) and rate-limiting. As a

result, the predominant steady-state intermediate in the

presence of physiological ATP concentrations is the

strongly bound AM.ADP state, making myosin-V a

high-duty-ratio motor.

It is important to recognize that both the duty ratio and

attachment lifetimes reflect enzymatic adaptations

required for specific biological functions, and that a high

duty ratio does not necessarily indicate that the actin-

attached intermediates have long lifetimes (see the ani-

mation in supplementary material for an illustration of

this principle). For example, myosin-V has a much greater

duty ratio than subclass-2 myosin-I proteins (Figure 2c),

yet the lifetime of the strong binding states of myosin-V

are significantly shorter than those of the low-duty-ratio

myosin-I isoform (Figure 2b). Myosin-V is better suited to

rapid transport, whereas it appears that subclass-2 myosin-

I proteins are designed for slow transport in actin mesh-

works or for tension maintenance [15,17].

Myosin-VI

Myosin-VI is apparently a two-headed molecule that does

not associate into filaments and that may play a role in

endocytosis and membrane trafficking [41]. It was the first

myosin discovered to move toward the pointed end of

actin filaments [42,43]. Like myosin-V, myosin-VI is a

high-duty-ratio motor as a result of its slow rate-limiting

ADP release [44]. Additionally, ATP binding is slow and

weak, resulting in a population of nucleotide-free AM

(rigor) state intermediates under physiological conditions,

which further increases the duty ratio [44].

Myosin kinetics and processivityA high duty ratio is necessary for continuous movement

by myosin motors. Myosin-I and most characterized

myosin-II isoforms are low-duty-ratio motors and must

work in ensembles of many motors (which generates a

high effective duty ratio) to sustain continuous sliding

(Figure 3a). This requirement has been demonstrated invitro by examining sliding as a function of protein

density [45–47]. Locally high concentrations of myo-

sin-II are created in cells by the assembly of myosin into

filaments and by incorporation of the filaments into

actin-containing structures such as stress fibers or sar-

comeres. Myosin-I isoforms are restricted to membranes

and the actin-rich cell cortex, but it is not known if

accessory proteins play a role in concentration of these

motors.

Myosin-V from vertebrates [28–31,32��,33��,34�,35�,36],

but not yeast [48], and myosin-VI [49,50] are highly

processive, which is generally agreed to mean that an

individual molecule can take multiple steps, each

coupled to a cycle of ATP hydrolysis, along an actin

filament track before dissociating. Both heads are

required for processivity. Each motor of a processive

two-headed myosin (in which the motor domains are

not independently processive) must have a high duty

ratio (>0.5) to increase the probability that at least one

motor domain is always attached to actin, or the motor and

transported cargo will diffuse away from its track. In

addition, there must be some form of communication

between the motor domains: at a minimum to bias the

position of lead head attachment, but perhaps coordinat-

ing the catalytic cycles.

Many models have been proposed to account for the

motility of two-headed processive myosins [28–31,32��,34�,35�,36,51,52,53��,54,55]. Most implicate coordination

of the ATPase cycle kinetics of the two heads. Although

head–head coordination is not a requirement for proces-

sivity, it has been observed for double-headed myosin-VI

[44] and has been shown to be Ca2þ-dependent [56].

We favor an alternating-site ‘hand-over-hand’ model [44]

for processive stepping in the presence of saturating ATP

(Figure 3b). The model predicts that myosin dwells

predominantly in a state with the trailing head strongly

bound to actin in the AM.ADP state, and the leading head

in equilibrium between a detached and an ‘engaged’

ADP.Pi state. The engaged head is bound to actin but

has not undergone its power-stroke. The trailing head

impedes the leading head from binding actin strongly and



Figure 3

ADP.Pi ADP

ADP

ADP.Pi

ADP.Pi

ATP

ADP.Pi

Pi

ATP

ADP.PiADP

ADP

Weak Strong

WeakStrong

Strong Strong

‘Engaged’ Strong

Weak Strong

(a) (b)


Proposed mechanisms for processive motion by myosins. (a) In the absence of accessory proteins that may tether the motor to the actin

filament, low-duty-ratio myosins must work in high concentrations to create a high effective duty-ratio and support sustained motility.

Top panel: a few low-duty-ratio motors cannot sustain continuous sliding, and the motor and cargo dissociate from actin. Bottom panel:

A high concentration of low-duty-ratio motors creates a high effective duty ratio, and the motors and cargo travel along the actin filament.

(b) Model for processivity by two-headed high-duty-ratio myosins. Myosin dwells predominantly in a state with the trailing head stronglybound to actin in the AM.ADP state, and the leading head in equilibrium between a detached and an ‘engaged’ ADP.Pi state. The engaged head

is bound to actin but has not undergone its power-stroke. The trailing head impedes the leading head from binding actin strongly and slows the

rate of Pi release. ADP release from the trailing head results in a conformational change that optimally positions the leading head for actin

attachment, allowing the lead head to bind actin strongly, begin its power stroke and eventually release Pi. ATP binding to the trailing head

dissociates it from actin, the leading head power stroke swings the trailing head forward and the previous leading head now becomes the

trailing head.



slows the rate of Pi release. ADP release from the trailing

head results in a conformational change that optimally

positions the leading head for actin attachment, allowing

the lead head to bind actin strongly, begin its power

stroke, and eventually release Pi. The conformational

change can be a rotation of the lever arm [31,33��,34�]or, with myosin VI, a more complex, larger-scale rearran-

gement that has yet to be determined [49,55]. At low ATP

concentrations (at which some structural experiments

have been performed), it has been proposed that the lead

head may release its products before the trailing head

dissociates [53��]. ATP binding to the trailing head dis-

sociates it from actin, whereupon the leading-head power

stroke swings the trailing head forward and the previous

leading head now becomes the trailing head. A defining

feature of this model is that the trailing head restricts the

lead head from progressing through the cycle.

Conclusions and future directionsThough the ATPase cycles of myosin motors are con-

served, the kinetics of the individual reactions defining

the cycle can vary dramatically and are optimized for

specific biological functions. It is generally believed that

the tail domains of myosin motors provide the source of

functional diversity. However, detailed kinetic analysis

demonstrates that modulation of the rate and equilibrium

constants defining the ATPase cycle can confer unique

and specific properties on a motor that allow it to accom-

plish a specific physiological task. Understanding the

kinetic mechanisms allows us to better define the poten-

tial cellular functions of the different myosin classes and

isoforms.

Future kinetic studies need to address the molecular

mechanisms of ATPase cycle regulation by associated

light chains [12,57–60], actin-binding proteins such as

tropomyosin, and post-translational modifications. It is

also critical to understand how external forces and loads

influence the rate constants of the ATPase cycle.

AcknowledgementsWe thank members of the De La Cruz and Ostap laboratories for helpfuldiscussions, and Lee Ligon and Chris Yengo for comments on themanuscript. EDLC is supported by grants from NSF (MCB-0216834)and a Scientist Development Grant from AHA. EMO is supported bygrants from NIH (GM57247) and an Established Investigator Awardfrom AHA.

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53.��

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Relating biochemistry and function in the myosin superfamily

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