Molecular motors designed for different tasks and to operate at different temperatures

.I. tkrm. Viol. Vol. 22, No. 6, pp. 367-373, 1997 (’ 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0306.4565/98 $19.00 + 0.00

PII: s03064565(97)ooo56-9

MOLECULAR

MOTORS

TASKS

AND

DESIGNED

TO OPERATE

FOR DIFFERENT

AT DIFFERENT

TEMPERATURES L. GAUVRY,

V. MOHAN-RAM,

C. ETTELAIE,

S. ENNION

and G. GOLDSPINK*

Department of Anatomy and Developmental Medicine,

Biology, Royal Free and University Hill Street, London, NW3 2PF, U.K.

Rowland

MOLECULAR MOTORS FOR DIFFERENT TASKS

Movement

is a basic

achieved force

by molecular

for movement

animals.

movement

duction

of ions

molecular

motors

systems.

associated

involved

with

Kinesins

out

the

animal

are

molecule, one member

of cell

crystal

to be responsible

the

of the protein

structures

systems

the

muscle

myosins.

myosins

cell shape

which and

The

myosins

a dimer and others consist

character-

others

are involved

producing

in deter-

this is generated to as subfragment

the sliding filaments.

by the myosin

one

catalytic

parallel,

Sl

which contripho-

should

direct

and

et

by

(Fisher

1995, 1996). In

of force and displace-

by a single myosin actin

al.,

discoideum myosin

Rayment,

measurement filament

has

interacting been

carried

with out

assay in which the force as

(Finer

by a technique

et al., 1994). From

the

of movement

it can be seen that the cyto-

skeletal

myosins

very

muscle

myosins.

are

ATPase ATP

cing slow changes 367

(Rayment

of the scallop myosin

well as the velocity can be estimated

they hydrolyse

be addressed.

resolution

(Xie et al., 1994) followed

called light trapping velocity

pectoralis

at a nominal

fragment

using an in vitro motility

of a long CI-

The three-

at 2.7 A and 1.9 A resolution

thus very economical * To whom correspondence

separately.

Dictyostelium

the

domain

a suspended

head, also referred

domain

from

ment generated

helix, part of which acts as a lever arm to which the essential (LCI and LC3) and regulatory (LC2) light and a globular

the

the three-

parts of the mol-

of the adult chicken

et al., 1995; Smith

The force for

tains binding sites for actin and adenosine sphate (ATP).

structure

2.8 A of

motor

of the actin

1 (Sl). This consists

of different

has been determined

head

in muscle

which is important

et al., 1993a,b) and

1993a,b). The X-ray structure

ways from the class

formation

over the myosin

of

as some do not exist as

which

several

et al., 1990; Holmes

ecule has to be characterised

cell movement.

sin filament

of

have been

which is a large molecule

structure

muscle has been determined

contraction

related

structure

proteins

(Rayment

are the

for myo-

a knowl-

family. Recently,

dimensional

of only the myosin

types

locomo-

requires

of contractile

smooth

with no tail or rod. The latter is required involves

dimensional

out that

of the cardiac,

in different

of a closely

for actin (Kabsch

case of myosin

the force for

be pointed

and

kinesin (Ku11 ef al., 1996; Sablin et al., 1996). In the

actin-related

that generate

It should

biology

for different

three-dimensional

determined

represent

motors

structure

et al., 1990) myosin

II or conventional

chains,

i.e.,

et al.,

to enable us to visual-

This approach

of the crystal

looked

They differ in several different

filaments

molecular

mitosis

which

molecular

graphics

tory requirements.

cell types

we have taken a compara-

of muscles which have evolved edge

and func-

in different 1994; Uyeda

and combined

computer

ise the myosin

along axons

there are at least twelve classes of myosin

mining

molecular

during

motor

cytoskeletal

tive approach

are the

in a variety

of chromosomes

locomotion.

skeletal

for cells.

which

of proteins

ised to date. Class II consists and

1994). In our laboratory

type and actin kinesins

motors

et al.. 1994; Spudich,

(Lee

in the trans-

former

of vesicles

the

using emerging

has shed light on the design

tion of these molecular

cells and

of the

technology

In this work we have, however,

myosins

molecular

and

are also believed

for the movement and meiosis. at

within

involved

the

in the movement

the movement

types.

are responsible

in and

Dyneins

is

cells and whole

RNA

motors

Over the past few years research

This

produce

of force include both tubulin

type motor

and

motors and

life.

that

of individual

of proteins

Eucaryotic

of

motors

Other molecular

movement the

property

College School of

much

slower

measurements

than

at a very slow rate as well as effective

in cell shape.

the

also show and

are

for produ-

L. Gauvry et al

368 The

use

of

a

slime

mould

model

Dictyostelium discoideum has allowed in vitro of Dictyostelium myosin cells devoid

using both

lytic fragments demonstrated

al.,

1995;

in native

have

still not

economical the muscle taining force.

in the transduction

of force

of contraction

is determined

activity of the myosin Muscle,

and

as an elastic

et al., 1996). However,

(Uyeda

for

Nevertheless,

within

In order

skeletal

development

the

skeletal

muscle

of

force.

muscle

were

taken

muscles

move

prey value.

economically

the survival

sample

(Chomczynski

expressed

in

extracted

the adaptation

and evolution

the resulting

using

Positive

recombinant

for the main-

skeletal

contracting than

isoform

1952 A.

movement.

V.

Hill

isoform

muscles

out

with many

(Hill,

1950). Thus

The molecular

is the

the

long

sarcomeres

series would need to have a slower intrinsic of shortening

is

et al., 1995). that

the

in

velocity

2b myosin

would be too fast for the muscles of large animals. If the 2b myosin was expressed, the very high stride frequency

would

the members

be unsustainable.

of the myosin

In vertebrates

heavy chain family are

coded for by separate genes. These are highly conserved but in the myosin Sl there are two variable regions

called

surface

loops

I and 2, which

by substituting ture

the distribution

hydrolysis

overlapping

myosin

into the crystal

subfragment

of charged

residues

acids which

1 (Fig.

differed

1). that

between

by differences

results

and struc-

1 revealed

loop

This was accentuated

amino

loop

using the Alchemy111 program

length of the loop. The fast type 2 isoform extra

site

and characterized.

and the surface

of the hypervariable

isoforms.

and

of the region pocket

the sequences

of chicken

Analysis

(Invitrogen).

heavy chain locus for

pocket

were isolated

binding

1 was visualized

to the pCRI1

system

clones were sequenced.

structure

the nucleotide

A PCR product

in the has two

in this loop being

longer than that of the slow. A progressive

shift of

an identified charge motif around the loop was observed from fast to slow to embryonic myosin Sl clones.

Positional

noted.

These

conservation

findings

imply

of charge that

was also

this flexible

loop

does serve a function.

form

the junctions between the three tryptic fragments (25, 50 and 20 kDa), localised in the N-terminal region of the coding part of the gene. Loop 2 is implicated in the actin binding thought to have a motor-velocity

cloning

binding

from three isoforms

by the 2x muscles

the nucleotide

is

mam-

The 2b myosin followed

pointed

but

of smaller

a TA

primer.

From this study the myosin

species in re-

the 2a. In human

of large animals

using the sense

vector

amongst

muscles

were amplified

(5’-ACAGAGAAAACCAGTCCATC-3’)

1 myosin

or for slow repetitive

in human

cDNAs

Exons 6 to 8 of

230bp was ligated

pro-

(Gibco,

a highly

isoforms.

antisense

contractions

in the skeletal

region between

of approximately

main fast types are 2x and 2a (Ennion In

reverse transcriptase

and the precedent

the slow type

mals such as the rat and mouse.

muscles

to the heavy

primer

the

was syn-

oligonucleotide primer was of the R-cardiac 3’ end 8 exon (5’chain

rapid, powerful

to body size. The fast 2b myosin

which is faster

designed

conserved

a range of contrac-

to produce

are also observed

the fastest

antisense

cDNA

GAAGCGGGAGGAGTTGTCGTTC-3’)

slow, economical

expressed

using MMLV An

extraction

1987). From

first strand

in

whereas

not

RNA

skeletal from each

single-step

factor

to be met. The fast type 2 isoforms

of posture

the

and Sacchi,

is also an important

movements

Differences

total

different

myosin

for example

expressed

has

using

and

the

1. Samples

was extracted

to

family to enable

tile demands

lation

predators

heart

included

loop

ability

are adapted

tenance

or avoid However,

Sl

the

isms have necessitated

duces

The ability to move

of many species. Such survival mechan-

of the myosin

which

the

muscle

BRL).

survival

from

in differ-

the

of the dog. RNA

method thesised

to capture

expressed

of the predominant

of

myosin

a given muscle fibre is one of the main determinants characteristics.

part

of force devel-

motor

and main-

the part

for

of its contractile rapidly

we cloned

site and the hypervariable

The type of molecular

obvious

to study the myosin

ent fibres ATPase

family there is a range of velocities opment.

el-

the velocity

myosin

mammalian

of

DESIGN OF MOLECULAR MOTORS EXPRESSED IN DIFFERENT FIBRES WITHIN THE SAME MUSCLES

by the specific ATPase

rapid

loop1 is as-

is therefore

is for developing

Sl head (Lowey et al., 1993).

particularly

designed

site and

et

(Itakura

et al., 1996;

Uyeda

1994). The hypervariable

with the ATPase

interest as this may determine the ATPase activity and hence the velocity of shortening and also how

et al., 1997). The lever arm is important

Kurzawa

is

head

tion (Spudich, sociated

and proteo-

a role for the light chains et

as

(Lee et al.,

recombinant

of the myosin

a/., 19%; Waher

ement

directly

of the wild type isoform

1994). Studies

such

the expression

while loop I is modulating func-

MOLECULAR MOTORS IN FISH MUSCLE DESIGNED TO OPERATE AT DIFFERENT TEMPERATURES

Fish point

provide that

good

different

examples isozymes

to

illustrate

(protein

the

isoforms)

Molecular

motors

for different

369

tasks

Panel A

Fast

E K K KE +-++ slow E R S KK ++ -+

EATSGKMQ + DQTPGK _

+

Panel B

canine slow loop

canine fast loop 1

Fig. 1. The deduced amino acid sequence alignment of loop 1 from canine chains and the charge residues from loop I are shown in Panel A. Deduced of the canine fast and slow loop I after substitution of different residues structure of Rayment el al.. (1993a. b) using the Alchemy III program

have evolved ranging

from

some species species range +6”C above these during

FATI

to operate

species occupy

Different

sub-zero that

at

at the poles

of fish are restricted (Somero this latter This

evolution

the

temperatures.

thermal

live in geothermal

e.g. Antarctic

fish.

different

well-defined

niches

to 45°C springs.

to a narrow

fish are restricted

for

Most thermal

and activity

heavy

three-dimensional structure from the fast chicken loop are shown in Panel B.

other and

proteins

to

thermal

acquire

stability

the

for that

temperature. Some fish, such as carp, can adapt to a wide range of temperatures by expressing different sets of myosin

heavy chains at warm and cold tem-

to

perature

(Gerlach

and DeVries, 1967). Warming temperature results in death for

Watabe

et al., 1995; Imai et al., 1997) as well as

type

of adaptation

and has involved

to -1.5

enzymes

appropriate

fast and slow myosin

I

has

occurred

the alteration

of

different

light

1990). Indeed, between

et al., 1990; Ennion

chains

(Crockford

and

higher activity

and thermal

- - - SK-

‘FATI

- GSLEDQI I 20 I - - - SK- - GSLEDQI

I AANPL L ESYGNAKTV I I 30 40 I I I AANPLLESYGNAKTV

ANTARCTIC

FATI

SVSGPKRDA-

- - - SK-

- GSLEDQI

I AANPL

L ESYGNAKTV

TREM. BERNAClll

FATI

SVSGPKRDT-

- - - SK-

- GSLEEQI

I AANPL

LESYGNAKTV

PLACELET

- - - SK-

- GSLEDQI

FATI

SVGGPKRDTAVSGGKKEAEP-

FATI

AAL GAKKFig. 2. Deduced

- SKMQGSLEDQI

AEPTPGKMQGSL

EDQI

Johnston,

there seems to have been a trade off

SVSGPKRDAI IO I SVSGPKRDA-

FATI

ef a/., 1995;

stability

ROCK COD PENNELLII

ICEFISH

I AANPLLEAYGNAKTI

SCULPIN

I AANPL

L ESYGNAKTV

TROUT

VAANPL

L EAYGNAKTV

BLACK SIDED HAWK

amino acid alignment of the region overlapping the loop 1 myosin different fish species (Antarctic, sculpin, trout and tropical fish).

of the

heavy chain

from

370

L. Gauvry

et al. ANTARCTIC ROCK COD TREM. BERNACII I PENNELLII

I

I

PLACELET ICEFISH SCULPIN TROUT BLACK SIDED HAWK

12.4 I

I

12

10

I

I

I

I

4

2

I

Fig. 3. Phylogenetic tree deduced from the region overlapping loop 1 myosin heavy chain for different fish species (Antarctic, sculpin, trout and tropical fish). enzyme

systems

involved

1975). The main

(Johnston

features

and Goldspink,

which

characterises

(5’-AAGTATGACAAAATTGAGGA-3’)

fish

8

muscle adaptation to a cold habitat are the increase of thermal instability of the whole myosin (Connell,

and

1960,

TAGAAGA-3’

1961; Ogawa

between habitat

temperature

Johnson twitch than

et

al.,

the maximum and

(Penney

Johnston,

activation warm

1993)

tension

and Goldspink,

1991). The

and relaxation

water

species

et

motors

to work at different

plified

by the myosin

and temperate

time

and

design

Sl from

fish has therefore

TA cloning

for

isolated

from

tropical

species

study,

Antarctic

described

overlapping loop

1 region

different

the

classification there

our atten-

loop

and

fish,

hawk)

amplified.

was RT

PCR

temperate

and

approach

A fragment

binding

trout, with

were as

heavy

of 300 bp

site and

the surface

fish species

(Antarctic

sculpin This

and

was

primers

black

carried

from

the

sided out by exon 4

were cloned

with

in pCR

kit from

fish species based

the

on

ROCK COD

TREM. BERNACII PLACELET

I PENNELLII

ICE FISH

SCULPIN TROUT

Fig. 4. shows the distribution

and

loop

have

sequence

comparison

differences

in

and size

is variable

and ranges

suggests

the of

compared

of and

-

SK + SK + SK + SK +

1

by The

demonstrated

charge.

The

total

to fish species (Fig. 4)

(+2) (i.2) (+2)

(+I) (+2) of loop 1

compared

1 from mammalian

c+21

SKWl t

residues

jbrsteri.

hybridisation.

for fish fast muscle of loop

.AKK AEPTPGKMK t tt of the charge

the and

+ 1 to + 2. Also the total charge

important

to the total charge

PKRDA + + -. PKRDA ++PKRDT ++PKRDT ++GKKEAEP

from

of fish

characterised

loop total

type the

coriiceps

Paracirrhites

been

by in situ

according

from

3). The

region

origin

from

also

blot

(Fig.

between

been

overlapheavy chain

of Nototheniu

fast myosin

clones

is more

the

have

fast myosin

Northern

+t

BLACK SIDED HAWK

2.1 vec-

in Fig. 2 and

tree

phylogenetic

Sequences

the tropical

charge

is shown

phylogenetic

is a correlation

Antarctic These

1 of the myosin

Total charge ANTARCTIC

two

Invitrogen)

of the coding-sequence loop

deduced

that

of the dog myosin

from different

notothenoid using

from in

tropical

products

the same

previously.

the ATP

PCR

and related

employing

for the characterisation chains

The comparison

species. the fish myosin

initiated

(5’-TCATCAGCTGA-

sequenced. ping the surface

activity

exon

S-AGCTCCAGTCAGCTTG-

tor (original

tion. For

and

198 1;

as exem-

attracted

cDNA primers

product

in cold

Antarctic,

strand

myosin

TA-3’). PCR

of molecular

temperatures

first

the

Johnston,

of the ATPase

1975). The

al.,

half

are longer

(Johnson

1991) as well as an increase (Johnston

and

a

conserved

a correlation

produced

and

(S-AAAACGAGAGGAGTTGTCATTCCT-3’)

fast

Molecular

myosin

( + 1) and is identical

heavy chain

charge

from

slow isoform.

seems

to

charge

of loop

be

no

Interestingly,

correlation

1 and

fish compared

and

myosin

tropical

the

heavy chain loop of the instability

temperatures

total

cold temperatures warm temperatures.

for

of the trout,

4) or mammalian

I. The decrease

at different

371

decrease

temperature.

to the

of the myosin.

instability.

mutagenesis ameters

There

studies

nature

1 influences

motility

It

has been

control

suggested

the veiocity

binding appears

ing the ATP

hydrolysis

selective cleavage tion

but

(Bobkov

it

that

does

Mg”

-ATPase

smooth

affect

using

isoforms

release.

have suggested

determination

data

of

derived

from et al.,

1996). We propose that the hypervariable loop 1 functions as an electrostatic latch, the opening and closupon charge-relationships

as well as the length

of the loop.

the number,

and

residues

polarity

that are formed.

a correlation

between

velocity

positive

1 (Kelley

to negative

canine

of charged

of electrostatic

Others authors

and a higher actin-activated size of loop

It is logical that

organisation

will affect the strength

actions

inter-

have shown

of actin translocation Mg2 +-ATPase

with the

et ul., 1993). The ratios residues

within

slow, fast and embryonic

the loop

isoform

of for

show that

this is true: the type 2 loop has an overall charge of + 1 whereas embryonic +2

and

mammals, between from

the

isoforms +2.5

contracting

possess

charges

of the loop

fast and slow myosin

1 increases

not

shown).

within

with residues

the structure

and

in the surrounding

the ease by which the powerstroke the

speed

at

which

ADP

par-

(Uyeda

imposes

et

ul.,

constraints

since all myo-

and bind to actin with similar

constraint. The decrease of temperature to cause local structural changes in the Sl

fragment

and a more

rigid Sl-nucleotide

structure

(Papp et cd., 1992).

their

of

of charge interaction

Sl. that determines occurs

can be ejected

binding pocket. The correlation different parts of the molecule,

CONCLUSION

and

from

The question

that

needs

does the structural bution

within

another

governing

is that part

using

loop

forms

would

of the myosin provide

important

sition

the structure

to isoform in

However, structure

this

by

may

and thus change from

take

the comparative

Nature

data

the active

has designed

A with

studies

myosin

iso-

to which

we

1 mutants

specific the

molecular

the function

tinct

Kinetic

different

Loop site

affect

of the myosin

interact

of loop 1 and its compo-

activity.

vitro

proprieties?

head.

from

distri-

1 contribute

1 may

loop

1 mutants

can correlate generated

loop

contractile

the

is how

and charge

the hypervariable

to mechanism possibility

to be addressed

composition

three-dimensional motor

of other

site. Therefore approach molecular

tasks and to work over different

can be

mutagenesis.

and motors

as a whole regions

we prefer

disto

to see how for different

ranges of tempera-

ture.

Acknowledgements--This work was funded by grants from NERC, the Kennel Club and a Dowager Countess Eleonor Peel Fellowship.

We

not only is it the length

the loop, but also the specific distribution clusters

that

kinetic

of

as in

heavy chain isoforms

(data

coriiceps

that

1 and

In fish species,

the total charge

propose

type

net positive

respectively.

Nototheniu

therefore

slow

myosin

structure

myosin

The

release

(Perreault-Micale

ing of which is dependent

the

of these insertions

the motor

ADP

authors

in the

activity

muscle

by affect-

it inhibits

not

1 is involved

loop

I may

loop

motor

from directed

loop 1 by tryptic diges-

er al.. 1996). Other

that

surface

rate or ADP

of surface

has demonstrated

function

that

of the myosin

of

1994). The presented on the localisation

is evidence

on Dictyostelium

of loop

and

to

compared to those living in This could be related to the

sins must recognise DISCUSSION

show that there has been a

on the size of the loop in fish adapted

thermal the

in size of

fish could be related

for different tasks

there

1 is smaller

to that

fish (Fig.

1 in the Antarctic

increase

between

environmental

sculpin

to total

our results

the size of the loop

the Antarctic

loop

From

motors

thus

the

of charge between the fact that the

loop is highly flexible, and that the slowest isoforms have the greatest overall positive charge suggest that such a mechanism could exist, Comparative studies of the loop 1 region from fish species living

REFERENCES

Bobkov, A. A., Bobkova, E. A., Lin, S. H. and Reisler, E. (1996) The role of surface loops (residue-204-216 and residue-627-646) in the motor function of the myosin head. Proceedings of the Academy of Sciences oj the United States ofAmerica 93, 2285-2289. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anulytical Biochemistry 162(I). 156-159. Connell, J. J. (1960) The relative stabilities of skeletal muscle myosins of some animals. Biochemistry Journal 75, 530-538.

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