Crystal

structure

of

the

ferritin

from

the

hyperthermophilic

archaeal

anaerobe

Pyrococcus

furiosus

The

crystal

structure

of

the

ferritin

from

the

archaeon

hyperthermophile

and

anaerobe

Pyrococcus

furiosus

-LRB-

PfFtn

-RRB-

is

presented

While

many

ferritin

structures

from

bacteria

to

mammals

have

been

reported

until

now

only

one

was

available

from

archaea

the

ferritin

from

Archaeoglobus

fulgidus

-LRB-

AfFtn

-RRB-

The

PfFtn

exhibits

the

point-group

symmetry

that

is

characteristic

of

most

ferritins

which

suggests

that

the

symmetry

found

in

the

previously

reported

AfFtn

is

not

a

common

feature

of

archaeal

ferritins

Consequently

the

four

large

pores

that

were

found

in

AfFtn

are

not

present

in

PfFtn

The

structure

has

been

solved

by

molecular

replacement

and

refined

at

resolution

to

R

and

Rfree

The

ferroxidase

center

of

the

aerobically

crystallized

ferritin

contains

one

iron

at

site

A

and

shows

sites

B

and

C

only

upon

iron

or

zinc

soaking

Electron

paramagnetic

resonance

studies

suggest

this

iron

depletion

of

the

native

ferroxidase

center

to

be

a

result

of

a

complexation

of

iron

by

the

crystallization

salt

The

extreme

thermostability

of

PfFtn

is

compared

with

that

of

eight

structurally

similar

ferritins

and

is

proposed

to

originate

mostly

from

the

observed

high

number

of

intrasubunit

hydrogen

bonds

A

preservation

of

the

monomer

fold

rather

than

the

assembly

appears

to

be

the

most

important

factor

that

protects

the

ferritin

from

inactivation

by

heat

Introduction

Ferritin

is

a

protein

involved

in

metal

homeostasis

by

reversible

storage

of

iron

and

presumably

also

in

protection

against

oxidative

stress

by

scavenging

reactive

oxygen

species

It

is

a

small

protein

of

approximately

kDa

and

its

main

structural

motif

is

a

bundle

of

four

parallel

α-helices

and

a

fifth

short

α-helix

tilted

towards

the

bundle

axis

and

masking

the

one

end

of

the

cylindrical

bundle

The

functional

ferritin

molecule

is

an

approximately

kDa

hollow

spherical

assembly

of

subunits

with

outer

and

inner

diameters

of

and

nm

respectively

The

ferritin

agglomerate

may

be

a

homopolymer

or

in

higher

eukaryotes

a

heteropolymer

consisting

of

homologous

subunits

designated

as

H

M

and/or

L.

Ferritin

subunits

except

the

L-type

subunit

incorporate

a

ferroxidase

center

-LRB-

FC

-RRB-

that

consists

of

a

μ-oxo

bridged

dinuclear

iron

group

and

is

responsible

for

iron

oxidation

Additionally

a

third

iron

site

is

observed

in

the

subunits

of

some

bacterial

ferritins

-LSB-

-RSB-

The

binuclear

iron

cluster

of

the

FC

and

the

third

iron

site

are

referred

to

as

A

B

and

C

iron

sites

respectively

Some

ferritins

also

called

bacterioferritins

-LRB-

BFRs

-RRB-

enclose

heme

groups

that

are

located

between

homodimeric

subunit

pairs

and

may

be

involved

in

electron

transfer

-LSB-

-RSB-

The

homologous

protein

DNA

protection

during

starvation

-LRB-

DPS

-RRB-

and

also

called

a

small

ferritin

is

a

spherical

assembly

with

smaller

dimensions

-LRB-

outer

and

inner

diameters

of

and

nm

respectively

-RRB-

and

iron

storage

capacity

-LRB-

iron

atoms

vs.

in

ferritin

-RRB-

than

ferritins

-LSB-

-RSB-

While

iron

is

believed

to

be

the

main

cationic

substrate

of

ferritins

zinc

accumulation

in

ferritin

has

also

been

reported

-LSB-

-RSB-

Ferritins

occur

in

a

wide

variety

of

organisms

from

prokaryotes

to

mammals

In

humans

their

function

is

related

to

iron

deficiencies

or

iron-overload

disorders

such

as

thalassemia

sickle

cell

anemia

hemochromatosis

and

pulmonary

hemosiderosis

-LSB-

-RSB-

Understanding

the

mechanism

of

the

action

of

ferritin

will

be

of

great

value

in

the

treatment

of

iron-related

diseases

Besides

its

medical

relevance

this

knowledge

is

also

of

fundamental

scientific

interest

especially

in

case

of

anaerobes

To

date

an

insight

into

the

iron-uptake

reaction

has

only

been

achieved

for

aerobic

organisms

In

these

organisms

Fe

-LRB-

II

-RRB-

ions

are

oxidized

possibly

together

with

phosphate

oxoanions

and

stored

inside

a

ferritin

shell

as

a

soluble

Fe

-LRB-

III

-RRB-

mineral

believed

to

be

a

ferrihydrite

mineral

with

approximate

composition

Ferric

ions

from

the

core

can

be

reduced

and

released

from

the

ferritin

molecule

when

needed

by

a

cell

Nevertheless

the

chemical

nature

of

the

physiological

reductants

for

iron

release

and

in

the

case

of

anaerobes

that

of

the

physiological

oxidants

for

iron

uptake

have

not

yet

been

established

Crystal

structures

of

ferritins

from

bacteria

fungi

plants

insects

and

vertebrates

are

available

whereas

those

from

archaea

are

limited

to

a

ferritin

from

the

sulfate-reducing

anaerobe

and

thermophile

Archaeoglobus

fulgidus

-LRB-

AfFtn

-RRB-

-LRB-

Protein

Data

Bank

PDB

codes

and

-RRB-

-LSB-

-RSB-

and

a

DPS

protein

from

Halobacterium

salinarum

-LRB-

PDB

codes

and

-RRB-

-LSB-

-RSB-

In

AfFtn

a

novel

point-group

symmetry

of

the

assembly

was

found

which

is

unusual

for

ferritins

and

was

only

known

previously

in

assemblies

such

as

the

DPS

proteins

-LSB-

-RSB-

and

also

in

the

ferritin

from

the

bacterium

Listeria

innocua

-LSB-

-RSB-

As

a

result

four

outsized

triangular

pores

about

wide

are

present

in

the

protein

shell

of

AfFtn

-LSB-

-RSB-

Pyrococcus

furiosus

is

a

strict

anaerobe

and

hyperthermophilic

archaeon

living

optimally

at

°C

It

was

isolated

from

the

hot

marine

springs

off

the

beach

of

Porto

di

Levante

in

Vulcano

Italy

P.

furiosus

is

a

fermentative

heterotroph

that

grows

on

starch

maltose

peptone

and

yeast

extract

and

produces

acetate

alanine

and

The

expression

of

the

native

ferritin

from

P.

furiosus

-LRB-

PfFtn

-RRB-

has

been

confirmed

by

N-terminal

sequencing

of

the

purified

protein

and

the

recombinant

ferritin

has

been

overproduced

in

Escherichia

coli

in

order

to

afford

extensive

biochemical

studies

-LSB-

-RSB-

Herein

we

present

the

structure

of

PfFtn

refined

at

resolution

In

contrast

to

the

previously

reported

first

structure

of

an

archaeal

AfFtn

the

functional

PfFtn

has

the

typical

point-group

symmetry

observed

in

other

ferritins

and

BFRs

There

are

no

four

large

pores

such

as

were

found

in

AfFtn

Instead

the

characteristic

threefold

and

fourfold

channels

are

present

in

PfFtn

The

extreme

thermostability

of

PfFtn

which

in

vitro

can

withstand

day

of

incubation

at

°C

has

been

analyzed

at

the

structural

level

in

comparison

with

structurally

related

ferritins

Although

P.

furiosus

grows

at

the

highest

temperature

°C

among

the

organisms

used

in

thermostability

analysis

our

results

suggest

that

the

ferritin

from

Thermotoga

maritima

-LRB-

TmFtn

-RRB-

which

has

an

optimal

growth

temperature

of

°C

may

be

even

more

thermostable

than

PfFtn

The

hyperthermal

stability

of

PfFtn

and

other

ferritins

appears

to

result

mainly

from

the

preservation

of

the

monomer

fold

rather

than

the

assembly

owing

to

a

high

number

of

intramolecular

hydrogen

bonds

between

main-chain

atoms

and

between

main-chain

and

side-chain

atoms

The

FC

stability

was

analyzed

by

electron

paramagnetic

resonance

-LRB-

EPR

-RRB-

Without

further

experiments

the

lack

of

FC

iron

sites

B

and

C

in

nonsoaked

PfFtn

crystals

obtained

does

not

allow

any

conclusions

supporting

or

contradicting

the

FC

as

a

stable

catalytic

cofactor

Materials

and

methods

Protein

production

and

crystallization

PfFtn

was

obtained

and

purified

as

previously

described

-LSB-

-RSB-

Ferritin

iron

loading

prior

to

crystallization

was

performed

by

adding

a

freshly

and

anaerobically

prepared

aqueous

solution

of

mM

iron

sulfate

containing

HCl

to

a

solution

of

apoferritin

in

mM

N

-LRB-

-RRB-

piperazine-N

ethanesulfonic

acid

-LRB-

Hepes

-RRB-

pH

under

aerobic

conditions

Crystals

of

PfFtn

were

produced

using

the

hanging

drop

vapor

diffusion

method

at

room

temperature

-LSB-

-RSB-

The

crystals

used

for

data

collection

were

grown

against

ml

of

M

ammonium

sulfate

reservoir

solution

with

drops

containing

μl

of

mg

ml

protein

solution

in

mM

Hepes

pH

and

an

equal

amount

of

the

reservoir

solution

The

crystals

of

the

as-isolated

ferritin

containing

approximately

Fe

per

grew

in

weeks

and

those

loaded

with

approximately

Fe

atoms

per

prior

to

crystallization

grew

in

months

The

crystals

exhibited

an

irregular

pyramidal

shape

with

base

side

and

height

between

and

μm

The

iron

content

of

as-isolated

ferritin

was

determined

with

ferrene

as

previously

described

-LSB-

-RSB-

by

measuring

the

absorption

of

the

iron

ferrene

complex

at

nm

ε

M

cm

Iron

soaking

of

ferritin

crystals

was

performed

by

transferring

them

to

a

drop

of

a

solution

with

composition

mM

mM

glycerol

and

M

ammonium

sulfate

for

min

Zinc

soaking

with

was

carried

out

in

a

similar

fashion

with

the

exception

that

dithionite

-LRB-

-RRB-

was

omitted

from

the

solution

X-ray

data

collection

Cryoprotecting

conditions

consisted

of

briefly

dipping

the

crystals

into

a

modified

crystallization

solution

containing

glycerol

in

the

case

of

the

as-crystallized

and

Fe-loaded

crystals

and

glycerol

in

the

case

of

the

Fe-soaked

and

Zn-soaked

crystals

prior

to

flash-freezing

at

°C

in

a

nitrogen-gas

stream

using

an

Oxford

Cryosystems

low-temperature

device

Diffraction

data

from

an

as-crystallized

an

Fe-soaked

and

a

Zn-soaked

crystal

of

as-isolated

PfFtn

as

well

as

from

a

loaded

PfFtn

crystal

with

approximately

Fe

per

were

collected

at

the

ESRF

beamline

from

flash-frozen

single

crystals

at

°C

Two

datasets

were

collected

from

the

Zn-soaked

crystal

the

first

at

the

Zn

K-edge

-LRB-

keV

Å

Zn

peak

-RRB-

as

determined

from

a

fluorescence

scan

and

the

second

-LRB-

keV

Å

Zn

low-E

rm

-RRB-

on

the

low-energy

side

of

the

absorption

edge

The

diffraction

images

were

integrated

with

the

program

MOSFLM

-LSB-

-RSB-

The

processed

data

were

scaled

merged

and

converted

to

structure

factors

using

the

program

suite

-LSB-

-RSB-

Data-processing

statistics

are

summarized

in

Table

Table

diffraction

data

collection

and

processing

statisticsBeamlineESRF

CCDMAR

CCDMAR

CCDMAR

CCDMAR

soak

Zn

soak

Zn

low-E

rmWavelength

-LRB-

Å

-RRB-

Space

group

cell

-LRB-

Å

-RRB-

Resolution

range

-LRB-

Å

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Unique

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Completeness

-LRB-

overall

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

I/σ

-LRB-

I

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Anomalous

completeness

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Anomalous

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Estimated

Boverall

-LRB-

Å2

-RRB-

Values

in

parentheses

refer

to

the

last

resolution

shell

Structure

determination

of

as-isolated

and

Fe-loaded

PfFtn

Previously

-LSB-

-RSB-

the

structure

of

PfFtn

was

solved

by

the

molecular

replacement

-LRB-

MR

-RRB-

method

using

the

program

PHASER

-LSB-

-RSB-

and

the

homologous

TmFtn

-LRB-

PDB

entry

-RRB-

as

the

search

model

The

sequence

of

this

amino

acid

ferritin

shares

a

amino

acid

sequence

identity

-LRB-

amino

acid

residues

-RRB-

with

that

of

PfFtn

-LRB-

amino

acids

-RRB-

and

the

only

insertions

or

deletions

occur

at

the

termini

of

the

subunit

chains

However

despite

the

success

of

the

MR

calculations

we

believed

that

because

of

the

low

resolution

of

the

diffraction

data

relatively

low

amino

acid

sequence

identity

with

the

search

model

as

well

as

the

complexity

of

the

structure

experimental

phase

information

would

be

needed

in

order

to

obtain

the

best

possible

structural

model

from

the

data

To

that

end

the

first

dataset

was

collected

at

a

long

wavelength

-LRB-

keV

Å

-RRB-

from

a

crystal

of

as-isolated

PfFtn

with

the

intention

of

using

S-SAD

in

phasing

whereas

a

second

dataset

was

collected

just

above

the

Fe

K-absorption

edge

-LRB-

keV

Å

-RRB-

from

an

Fe-loaded

PfFtn

crystal

with

the

aim

of

using

Fe-SAD

in

phasing

and

also

for

better

location

of

the

incorporated

Fe

atoms

However

neither

dataset

could

provide

the

desired

independent

phase

information

owing

to

complications

that

arose

from

radiation

damage

which

prevented

us

from

collecting

the

highly

redundant

dataset

required

for

application

of

the

S-SAD

method

to

the

first

dataset

as

well

as

the

lack

of

a

sufficiently

high

ordered

Fe

content

which

limited

the

usefulness

of

the

anomalous

dispersion

data

obtained

from

the

second

dataset

Therefore

the

structure

of

PfFtn

was

solved

by

repeating

the

MR

calculations

previously

described

-LSB-

-RSB-

with

the

new

as-isolated

dataset

using

the

standard

PHASER

protocol

as

implemented

through

the

graphical

user

interface

with

reflection

data

up

to

resolution

Previously

we

had

established

-LSB-

-RSB-

that

the

asymmetric

unit

of

the

PfFtn

crystal

structure

contained

monomers

arranged

as

The

search

model

used

was

half

of

a

TmFtn

constructed

from

the

PDB

coordinates

without

the

Fe

sites

and

three

such

models

were

searched

for

in

the

crystal

structure

of

PfFtn

The

MR

calculations

with

PHASER

gave

a

single

solution

Prior

to

model

rebuilding

electron-density

map

improvement

was

carried

out

both

by

using

Arp/wArp

-LSB-

-RSB-

in

atom

update

and

refinement

mode

as

well

as

by

using

DM

-LSB-

-RSB-

with

noncrystallographic

symmetry

-LRB-

NCS

-RRB-

averaging

In

this

way

the

initial

PHASER

figure

of

merit

of

for

the

full

resolution

range

of

the

dataset

was

improved

to

-LRB-

Arp/wArp

-RRB-

and

-LRB-

DM

-RRB-

Rebuilding

of

the

protein

chain

to

take

into

account

the

differences

in

sequence

and

chain

length

between

TmFtn

and

PfFtn

was

carried

out

with

the

program

TURBO

-LSB-

-RSB-

using

one

monomer

and

the

averaged

electron-density

map

obtained

from

the

DM

calculations

The

coordinates

for

each

of

the

remaining

monomers

were

obtained

from

those

of

the

first

by

applying

the

appropriate

NCS

transformations

followed

by

model

correction

with

TURBO

using

the

electron-density

maps

obtained

from

both

the

DM

and

the

Arp/wArp

calculations

In

these

maps

peaks

corresponding

to

Fe

occupation

of

the

A

sites

only

in

the

FC

were

observed

and

therefore

only

iron

sites

-LRB-

one

per

monomer

-RRB-

were

included

in

the

model

The

structure

of

the

Fe-loaded

PfFtn

was

also

determined

by

MR

with

PHASER

using

a

similar

procedure

however

the

Fo

density

map

as

well

as

an

anomalous

electron

density

map

calculated

with

MR

phases

showed

that

only

the

A

site

in

the

FC

was

occupied

by

Fe

atoms

Since

the

as-isolated

dataset

had

higher

resolution

and

better

quality

further

work

on

the

Fe-loaded

dataset

was

abandoned

Structure

refinement

of

as-isolated

PfFtn

Refinement

was

carried

out

with

the

program

REFMAC

-LSB-

-RSB-

using

medium

main-chain

and

weak

side-chain

NCS

restraints

between

the

dimers

in

the

asymmetric

unit

At

a

later

stage

a

translation

libration

screw

-LRB-

TLS

-RRB-

rigid-body

motion

refinement

-LSB-

-RSB-

was

made

prior

to

restrained

refinement

of

atomic

positions

and

thermal

motion

parameters

One

rigid

body

was

defined

for

each

of

the

independent

monomers

In

the

final

refinement

stages

a

total

of

solvent

molecules

were

located

with

Arp/wArp

-LSB-

-RSB-

On

the

basis

of

their

B

factors

observed

electron

density

and

interactions

with

neighboring

protein

residues

were

assigned

to

water

molecules

while

the

remaining

were

modeled

as

sulfate

ions

Individual

restrained

B

factors

were

refined

for

all

non-hydrogen

atoms

and

for

consistency

with

the

structure

refinements

of

the

Fe-soaked

and

Zn-soaked

crystals

-LRB-

see

below

-RRB-

only

tight

NCS

restraints

-LRB-

Å

positional

and

Å2

thermal

parameter

-RRB-

were

applied

to

all

the

protein

residues

involved

in

the

FC

metal

coordination

instead

of

defining

the

coordination

geometry

for

each

individual

protein

metal

bond

as

in

the

earlier

stages

of

refinement

The

final

values

of

R

and

Rfree

were

and

respectively

Neither

TLS

rigid-body

refinement

nor

full

NCS

restraints

-LRB-

i.e.

extending

over

the

whole

monomer

and

relating

all

the

chains

in

the

asymmetric

unit

-RRB-

were

used

in

the

final

refinement

as

they

were

seen

to

lead

to

higher

values

of

both

R

and

Rfree

Rfree

-LSB-

-RSB-

was

calculated

from

a

randomly

chosen

subset

of

the

data

containing

approximately

of

the

total

number

of

independent

reflections

Throughout

the

refinement

the

model

was

periodically

checked

and

corrected

with

TURBO

against

Fo

Fc

and

Fo

Fc

electron-density

maps

The

refinement

statistics

are

included

in

Table

The

maximum-likelihood

estimate

of

the

overall

coordinate

error

obtained

with

REFMAC

was

Å

For

all

independent

monomers

in

the

PfFtn

crystal

structure

only

the

first

residues

were

included

in

the

model

Although

some

electron

density

was

visible

beyond

it

was

not

possible

to

build

any

more

C-terminal

residues

into

it

and

it

is

likely

that

the

C-terminal

tail

is

disordered

in

all

the

independent

PfFtn

monomers

On

the

basis

of

a

B-value

comparison

with

the

protein

residues

directly

coordinating

the

iron

atoms

it

was

realized

that

at

least

out

of

the

metal

sites

were

not

fully

occupied

and

were

assigned

occupation

factors

of

-LRB-

in

one

case

-RRB-

In

addition

side-chain

atoms

could

not

be

seen

in

either

Fo

Fc

or

Fo

Fc

electron-density

maps

and

were

therefore

given

zero

occupancy

during

the

refinement

Table

and

structure

analysis

statistics

for

the

ferritin

from

Pyrococcus

furiosus

-LRB-

PfFtn

-RRB-

CrystalAs-isolated

as-crystallizedFe-soakedZn-soakedResolution

-LRB-

Å

-RRB-

No

of

reflections

in

work

set/test

Number

of

atoms

Protein

total

number

of

Protein

atoms

with

zero

Fe/Zn

Water

molecules

-LRB-

sulfate

ions

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mean

B

factors

-LRB-

Å2

-RRB-

Protein

main

chain

-LRB-

side

chain

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Fe/Zn

Water

molecules

-LRB-

sulfate

ions

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Root

mean

square

deviations

from

ideal

values

Bond

lengths

-LRB-

Å

-RRB-

Bond

angles

-LRB-

-RRB-

Mean

positional

error

-LRB-

Å

-RRB-

PROCHECK

Ramachandran

analysis

Total

no

analyzed

Residues

in

core

regions

no

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Residues

in

allowed

regions

no

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Residues

in

generous

regions

no

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Residues

in

disallowed

regions

no

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

PROCHECK

G

factors

Mean

-LRB-

range

-RRB-

-LRB-

to

-RRB-

-LRB-

to

-RRB-

-LRB-

to

-RRB-

Mean

-LRB-

range

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mean

-LRB-

range

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

structure

was

analyzed

with

PROCHECK

-LSB-

-RSB-

and

its

stereochemical

quality

parameters

were

within

their

respective

confidence

intervals

A

Ramachandran

-LSB-

-RSB-

ϕ

φ

plot

showed

that

of

the

non-glycine

and

non-proline

residues

-LRB-

-RRB-

were

found

to

lie

in

the

generously

allowed

regions

and

only

one

-LRB-

-RRB-

outlier

was

observed

All

these

residues

with

more

unusual

ϕ

φ

conformations

were

located

in

a

loop

region

containing

for

which

the

electron

density

was

generally

poor

The

final

atomic

coordinates

were

deposited

with

the

PDB

-LSB-

-RSB-

to

be

released

upon

publication

with

the

accession

code

Structure

determination

and

refinement

of

the

Fe-soaked

and

Zn-soaked

PfFtn

crystals

In

view

of

the

very

small

changes

in

cell

parameters

observed

in

comparison

with

those

of

the

as-isolated

crystal

the

structures

of

the

Fe-soaked

and

Zn-soaked

crystals

of

PfFtn

were

determined

directly

by

using

the

final

refined

coordinates

of

the

as-isolated

structure

omitting

the

iron

sites

and

the

solvent

atoms

Ten

cycles

of

rigid-body

refinement

were

first

carried

out

followed

by

a

TLS

rigid-body

motion

refinement

-LSB-

-RSB-

prior

to

restrained

refinement

of

atomic

positions

and

thermal

motion

parameters

At

this

stage

the

Fo

Fc

or

Fo

Fc

electron-density

maps

were

inspected

with

COOT

-LSB-

-RSB-

for

inclusion

of

the

FC

A

B

and

C

metal

sites

in

the

models

Because

of

the

complexity

of

the

metal

coordination

in

the

FC

the

low

resolution

of

the

diffraction

data

and

the

realization

that

some

of

the

FC

metal

sites

were

not

fully

occupied

-LRB-

see

Discussion

for

details

-RRB-

tight

NCS

restraints

-LRB-

Å

positional

and

Å2

thermal

parameter

-RRB-

were

applied

to

all

the

protein

residues

involved

in

the

FC

metal

coordination

instead

of

defining

the

cordination

geometry

for

each

individual

protein

metal

bond

In

the

final

refinement

of

the

Fe-soaked

crystal

structure

solvent

molecules

and

sulfate

ions

were

located

with

Arp/wArp

-LSB-

-RSB-

and

included

in

the

model

and

individual

restrained

B

factors

were

refined

for

all

non-hydrogen

atoms

On

the

basis

of

a

B-value

comparison

with

the

protein

residues

directly

coordinating

the

iron

atoms

it

was

realized

that

not

all

metal

sites

were

fully

occupied

all

the

B

sites

and

one

C

site

were

assigned

an

occupation

of

The

final

values

of

R

and

Rfree

were

and

The

refinement

statistics

are

listed

in

Table

The

structure

was

analyzed

with

PROCHECK

-LSB-

-RSB-

and

its

stereochemical

quality

parameters

were

within

their

respective

confidence

intervals

A

Ramachandran

-LSB-

-RSB-

ϕ

φ

plot

showed

that

of

the

non-glycine

and

non-proline

residues

ten

-LRB-

-RRB-

were

found

to

lie

in

the

generously

allowed

regions

and

only

eight

-LRB-

-RRB-

outliers

were

observed

With

three

exceptions

all

these

residues

with

more

unusual

ϕ

φ

conformations

were

located

in

a

loop

region

containing

for

which

the

electron

density

was

generally

poor

In

the

final

refinement

of

the

Zn-soaked

crystal

structure

solvent

molecules

and

sulfate

ions

were

located

with

Arp/wArp

-LSB-

-RSB-

and

included

in

the

model

and

individual

restrained

B

factors

were

refined

for

all

non-hydrogen

atoms

A

B-value

comparison

with

the

protein

residues

directly

coordinating

the

metal

atoms

combined

with

the

observation

of

a

mixed

iron/zinc

population

at

the

A

sites

-LRB-

see

below

for

details

-RRB-

led

us

to

postulate

a

mixed

-LRB-

Zn/Fe

-RRB-

occupancy

for

the

A

sites

which

was

modeled

as

a

Zn

atom

with

occupancy

Also

the

B

sites

were

modeled

as

partially

occupied

by

zinc

-LRB-

-RRB-

and

the

C

sites

as

partially

occupied

by

iron

-LRB-

-RRB-

The

final

values

of

R

and

Rfree

were

and

The

refinement

statistics

are

included

in

Table

The

structure

was

analyzed

with

PROCHECK

-LSB-

-RSB-

and

its

stereochemical

quality

parameters

were

within

their

respective

confidence

intervals

A

Ramachandran

-LSB-

-RSB-

ϕ

φ

plot

showed

that

of

the

non-glycine

and

non-proline

residues

nine

-LRB-

-RRB-

were

found

to

lie

in

the

generously

allowed

regions

and

only

seven

-LRB-

-RRB-

outliers

were

observed

With

two

exceptions

all

these

residues

with

more

unusual

ϕ

φ

conformations

were

located

in

a

loop

region

containing

for

which

the

electron

density

was

generally

poor

As

for

the

as-isolated

structure

neither

TLS

rigid-body

refinement

nor

full

NCS

restraints

were

used

in

the

final

refinement

of

the

Fe-soaked

or

Zn-soaked

structures

as

they

were

seen

to

lead

to

higher

values

of

both

R

and

Rfree

Rfree

-LSB-

-RSB-

was

calculated

from

a

randomly

chosen

subset

of

the

data

containing

approximately

of

the

total

number

of

independent

reflections

and

with

the

same

h

k

l

indices

as

for

the

as-isolated

data

These

test-set

reflections

were

not

chosen

in

thin

resolution

shells

However

while

it

is

true

that

the

have

very

high

NCS

in

all

three

refined

crystal

structures

the

Rfree/Rwork

ratios

are

typical

for

the

data

resolution

and

refinement

parameters

used

-LSB-

-RSB-

and

therefore

there

is

no

indication

that

the

more

simplistic

test-set

choice

may

have

introduced

a

bias

in

the

Rfree

values

Throughout

the

refinement

the

models

were

periodically

checked

and

corrected

with

COOT

-LSB-

-RSB-

against

Fo

Fc

and

Fo

Fc

electron-density

maps

Since

the

asymmetric

unit

of

both

structures

contains

monomers

and

each

monomer

contains

one

FC

with

three

metal

sites

a

total

of

metal

atoms

are

present

in

the

model

In

addition

side-chain

atoms

could

not

be

seen

in

either

Fo

Fc

or

Fo

Fc

electron-density

maps

for

the

Fe-soaked

structure

-LRB-

for

the

Zn-soaked

structure

-RRB-

and

were

given

zero

occupancy

during

the

refinement

The

final

atomic

coordinates

were

deposited

with

the

PDB

-LSB-

-RSB-

to

be

released

upon

publication

with

the

accession

codes

-LRB-

Fe-soaked

structure

-RRB-

and

-LRB-

Zn-soaked

structure

-RRB-

Results

and

discussion

Structure

of

the

PfFtn

monomer

The

PfFtn

monomer

exhibits

the

typical

four-helical

bundle

fold

which

is

encountered

in

ferritins

BFRs

DPS

proteins

and

rubrerythrins

According

to

the

DSSP

algorithm

-LSB-

-RSB-

the

four

helices

comprise

residues

-LRB-

helix

A

-RRB-

-LRB-

helix

B

-RRB-

-LRB-

helix

C

-RRB-

and

-LRB-

helix

D

-RRB-

A

short

C-terminal

α-helix

-LRB-

helix

E

residues

-RRB-

bordering

one

of

the

cylindrical

bundle

openings

followed

by

a

tail

without

any

secondary

structure

also

occurs

in

the

PfFtn

monomer

fold

-LRB-

Fig.

b

-RRB-

For

convenience

of

terminology

the

two

extremities

of

the

cylindrical

α-helical

bundle

will

be

further

referred

to

as

the

open

side

and

the

E-helix

sides

of

the

monomer

Fig.

Topology

diagram

of

the

ferritin

from

Pyrococcus

furiosus

-LRB-

PfFtn

-RRB-

b

Ribbon

diagram

of

PfFtn

showing

the

location

of

the

ferroxidase

center

sites

A

B

and

C

-LRB-

orange

spheres

-RRB-

near

the

center

of

the

four-helical

bundle

c

Superposition

of

the

PfFtn

Cα

chain

with

those

of

its

eight

closest

homologous

structures

In

b

and

c

the

molecule

colors

change

from

blue

to

red

in

the

N-terminal

to

C-terminal

direction

b

c

Prepared

with

PyMOL

-LSB-

-RSB-

The

PfFtn

monomer

is

built

of

hydrophobic

amino

acids

at

its

open

and

E-helix

sides

and

polar

and

hydrophilic

residues

in

the

middle

of

the

subunit

similarly

to

the

subunits

from

other

ferritins

The

open

side

of

the

monomer

contains

three

leucine

residues

-LRB-

and

-RRB-

two

phenylalanine

residues

-LRB-

and

-RRB-

and

whereas

the

E-helix

side

has

three

phenylalanine

residues

-LRB-

and

-RRB-

and

-LRB-

denoted

by

asterisks

in

Fig.

-RRB-

The

residues

defining

the

open

and

E-helix

sides

of

the

monomer

are

conserved

among

PfFtn

the

ferritin

from

E.

coli

-LRB-

EcFtnA

-RRB-

AfFtn

and

human

H

chain

ferritin

-LRB-

HuHF

-RRB-

in

terms

of

their

polarities

apart

from

which

in

other

ferritins

is

a

tyrosine

Fig.

acid

sequence

alignment

of

PfFtn

the

ferritin

from

Archaeoglobus

fulgidus

-LRB-

AfFtn

-RRB-

the

ferritin

from

Escherichia

coli

-LRB-

EcFtnA

-RRB-

and

human

H

chain

ferritin

-LRB-

HuHF

-RRB-

Asterisks

show

the

residues

around

the

open

and

E-helix

sides

of

the

monomer

A

B

and

C

denote

the

residues

coordinating

the

iron

sites

of

the

ferroxidase

center

-LRB-

FC

-RRB-

red

bars

highlight

the

main

residues

located

around

the

threefold

channels

and

gray

bars

those

around

the

fourfold

channels

The

alignment

was

prepared

with

ClustalW

-LSB-

-RSB-

The

central

part

of

the

subunit

comprises

the

negatively

charged

and

polar

amino

acids

involved

in

metal

binding

These

are

and

and

which

are

shown

in

Fig.

with

A

B

or

C

labels

to

denote

the

iron

site

they

coordinate

-LRB-

see

The

FC

-RRB-

In

addition

and

which

in

EcFtnA

-LSB-

-RSB-

indirectly

contribute

to

the

A

and

B

sites

via

hydrogen

bonds

to

and

are

also

oriented

towards

the

FC

in

PfFtn

It

has

been

noted

-LSB-

-RSB-

that

and

-LRB-

in

AfFtn

numbering

-RRB-

are

conserved

among

AfFtn

EcFtnA

and

HuHF

and

define

the

BC

loop

and

the

end

of

D

helix

respectively

In

the

case

of

PfFtn

only

is

conserved

while

the

two

glycine

residues

are

changed

into

and

A

surface

hydrophobic

pocket

at

the

PfFtn

dimer

interface

A

large

surface

pocket

largely

lined

by

hydrophobic

residues

was

located

at

the

PfFtn

dimer

interface

Interestingly

one

of

these

is

corresponding

to

Met

in

the

BFR

from

Desulfovibrio

desulfuricans

ATCC

-LSB-

-RSB-

which

binds

the

heme

group

Instead

of

a

heme

the

pocket

appears

to

be

occupied

by

a

disordered

string

of

water

molecules

-LRB-

Fig.

-RRB-

which

form

a

continuous

region

of

electron

density

This

feature

was

observed

for

all

the

PfFtn

structures

investigated

A

similar

situation

was

reported

by

Yu

et

al.

-LSB-

-RSB-

for

the

hydrophobic

cavity

of

human

interleukin

1β

Fig.

of

a

dimer

interface

in

the

as-isolated

PfFtn

structure

showing

a

difference

electron

density

feature

drawn

at

the

map

root

mean

square

-LRB-

RMS

-RRB-

level

which

results

from

a

disordered

chain

of

water

molecules

trapped

within

a

hydrophobic

pocket

centered

around

This

feature

is

observed

in

all

dimers

of

all

PfFtn

structures

investigated

The

bulk

of

the

PfFtn

dimer

is

represented

by

its

Cα

trace

-LRB-

blue-gray

-RRB-

The

side

chains

-LRB-

including

Cα

atoms

-RRB-

of

the

residues

in

both

monomers

are

represented

in

ball-and-stick

mode

and

are

colored

gold

The

view

is

down

the

noncrystallographic

twofold

symmetry

axis

of

the

dimer

looking

towards

the

inside

of

the

PfFtn

The

figure

was

prepared

with

DINO

-LSB-

-RSB-

Subunit

structure

comparison

In

order

to

compare

the

PfFtn

structure

with

the

structures

of

other

known

ferritins

a

protein

structure

comparison

against

the

PDB

using

the

secondary

structure

matching

-LRB-

SSM

-RRB-

tool

at

the

European

Bioinformatics

Institute

-LRB-

-RRB-

-LSB-

-RSB-

was

performed

This

resulted

in

hits

which

after

elimination

of

duplicate

protein

chains

were

seen

to

correspond

to

different

protein

structures

A

multiple

structural

alignment

was

then

carried

out

with

a

subset

of

eight

SSM

hits

listed

in

Table

and

shown

as

a

superposition

of

Cα

chains

in

Fig.

The

members

of

this

subset

were

chosen

such

that

the

percentage

of

their

aligned

residues

was

equal

to

or

greater

than

all

five

secondary

structure

elements

-LRB-

α-helices

-RRB-

were

aligned

the

Q

score

of

their

pairwise

SSM

alignment

with

PfFtn

was

greater

than

and

considering

only

one

crystal

form

of

each

different

protein

structure

from

each

organism

This

subset

includes

representatives

from

archaea

bacteria

and

eukarya

The

structural

alignment

showed

PfFtn

to

be

highly

similar

to

TmFtn

followed

by

AfFtn

and

EcFtnA

-LRB-

Table

-RRB-

The

deviations

occur

mostly

in

loop

regions

AB

loop

from

to

end

of

the

BC

loop

from

to

CD

loop

and

helix

D

up

to

and

DE

loop

from

to

-LRB-

in

PfFtn

numbering

-RRB-

Table

with

known

structure

most

closely

homologous

to

PfFtnPDB

IDProtein

nameSource

organismChain

lengthIdentity

-LRB-

-RRB-

Identity

SS-aligned

-LRB-

-RRB-

Q

scoreaRMSD

-LRB-

Å

-RRB-

-LRB-

-RRB-

Thermotoga

krqFerritinCampylobacter

eumFerritin

ECFTNAEscherichia

ferritinHomo

sapiens

fhaH

ferritin

mutantHomo

sapiens

mfrM

ferritinRana

rcgL

ferritinRana

PDB

Protein

Data

Bank

SS

secondary

structure

RMSD

root

mean

square

deviationaParameter

that

takes

the

alignment

length

and

the

RMSD

between

the

superimposed

atoms

into

account

and

varies

from

in

the

case

of

poor

matches

to

in

the

case

of

identical

proteins

Structure

of

the

PfFtn

and

comparison

with

AfFtn

A

highly

conserved

quaternary

structure

appears

among

all

known

ferritins

a

spherical

tetraeicosameric

assembly

with

point-group

symmetry

An

interesting

exception

to

this

rule

is

the

recently

reported

structure

of

the

AfFtn

-LSB-

-RSB-

which

exhibited

point-group

symmetry

normally

an

attribute

of

the

dodecameric

DPS

proteins

Even

though

PfFtn

and

AfFtn

are

highly

similar

archaeal

ferritins

-LRB-

and

amino

acid

sequence

identity

and

similarity

respectively

-RRB-

the

PfFtn

has

the

canonical

point-group

symmetry

instead

of

the

point-group

symmetry

found

in

the

AfFtn

Although

there

may

be

other

archaeal

tetraeicosameric

ferritins

with

point-group

symmetry

like

AfFtn

our

structure

clearly

suggests

that

it

is

not

a

feature

of

all

archaeal

ferritins

When

comparing

the

two

quaternary

structures

-LRB-

Fig.

-RRB-

it

can

be

seen

that

the

and

arrangements

in

PfFtn

and

AfFtn

result

from

a

different

organization

of

the

four

fundamental

units

hexamers

formed

by

three

dimers

around

a

vertical

turn

from

a

threefold

NCS

rotation

axis

The

arrangement

leads

to

the

appearance

of

four

outsized

triangular

pores

about

wide

in

the

protein

shell

of

AfFtn

-LSB-

-RSB-

which

are

absent

in

the

arrangement

of

PfFtn

and

all

other

known

structures

of

tetraeicosameric

bacterial

ferritins

Fig.

diagrams

of

the

PfFtn

and

AfFtn

I

View

down

a

threefold

axis

showing

one

fundamental

hexamer

II

View

rotated

by

approximately

about

a

vertical

axis

showing

the

different

hexamer

arrangement

which

leads

to

the

appearance

of

large

pores

in

AfFtn

The

figure

was

prepared

with

PyMOL

-LSB-

-RSB-

Threefold

and

fourfold

channels

The

eight

threefold

channels

are

formed

by

helices

C

and

D

from

three

adjacent

dimers

in

such

a

way

that

with

respect

to

the

hollow

tetraeicosameric

protein

shell

the

C-terminal

ends

of

helices

C

define

the

outer

entrance

to

the

channel

and

the

N-termini

of

helices

D

define

the

inner

entrance

-LRB-

Fig.

-RRB-

The

PfFtn

threefold

channel

is

lined

by

a

mix

of

hydrophobic

and

hydrophilic

amino

acids

and

at

the

outer

side

of

the

channel

and

in

the

middle

and

and

at

the

inner

entrance

of

the

channel

-LRB-

Fig.

-RRB-

In

general

and

similarly

to

EcFtnA

and

AfFtn

this

channel

is

less

hydrophilic

than

in

vertebrate

H

and

L

chain

ferritins

suggesting

a

rather

hydrophobic

threefold

channel

in

microbial

ferritins

in

comparison

with

vertebrate

ferritins

Nevertheless

when

compared

with

that

in

EcFtnA

and

AfFtn

the

threefold

channel

in

PfFtn

is

more

similar

to

that

of

HuHF

in

the

sense

that

is

it

more

negatively

charged

at

its

inner

entrance

and

more

positively

charged

in

its

central

region

-LRB-

Fig.

-RRB-

Fig.

view

of

the

threefold

and

fourfold

channels

in

PfFtn

AfFtn

EcFtnA

and

HuHF

The

exterior

of

the

shell

lies

on

the

left

side

and

the

inner

cavity

on

the

right

side

of

each

cartoon

as

shown

schematically

in

a

The

red

residues

correspond

to

acidic

Glu

and

Asp

residues

and

the

blue

ones

to

positive

Lys

Arg

and

His

residues

and

highlight

the

arrangement

of

positive

and

negative

amino

acids

along

the

channels

The

figure

was

prepared

with

PyMOL

-LSB-

-RSB-

On

the

basis

of

the

crystallographic

kinetic

mutagenesis

and

electrostatic

calculations

studies

on

HuHF

-LSB-

-RSB-

the

threefold

channels

were

proposed

to

be

involved

in

the

entry

of

iron

ions

into

the

ferritin

molecule

The

inhibition

of

iron

uptake

in

HuHF

upon

and

substitutions

-LSB-

-RSB-

and

the

coordination

of

by

and

-LSB-

-RSB-

have

been

observed

in

this

protein

The

inner

narrow

entrance

of

the

threefold

channel

in

PfFtn

resembles

that

of

HuHF

suggesting

that

a

similar

mechanism

is

possible

in

PfFtn

Nevertheless

the

mutations

of

and

also

influenced

the

iron-binding

capacity

of

HuHF

and

inhibited

the

binding

of

different

metals

by

the

channel

to

different

extents

The

different

amino

acid

arrangements

in

the

threefold

channel

of

the

various

ferritins

suggests

the

possibility

of

different

iron

incorporation

mechanisms

The

six

fourfold

channels

formed

around

the

fourfold

symmetry

axes

of

the

PfFtn

are

lined

from

the

outside

to

the

inside

of

the

protein

shell

by

and

-LRB-

Fig.

-RRB-

Owing

to

a

nozzlelike

shape

formed

by

the

PfFtn

fourfold

channel

is

longer

-LRB-

approximately

Å

-RRB-

than

its

counterparts

in

EcFtnA

-LRB-

approximately

Å

-RRB-

and

HuHF

-LRB-

approximately

Å

-RRB-

In

PfFtn

in

the

DE

loop

which

forms

an

outer

gate

to

the

fourfold

channel

occurs

in

place

of

EcFtnA

or

HuHF

making

this

gate

more

polar

and

charged

in

PfFtn

In

addition

the

DE

loop

and

its

amino

acids

are

somewhat

oriented

outwards

from

the

fourfold

channel

resulting

in

a

hourglass

shape

of

this

channel

which

is

less

strongly

developed

in

EcFtnA

and

even

less

so

in

HuHF

where

the

pore

has

an

almost

cylindrical

shape

The

outer

entrance

of

the

PfFtn

fourfold

channel

is

polar

and

hydrophilic

the

region

inside

the

channel

is

nonpolar

and

the

inner

entrance

is

polar

again

The

fourfold

channels

in

EcFtnA

have

a

similar

arrangement

of

polar

residues

at

both

channel

entrances

and

are

nonpolar

in

the

middle

while

in

HuHF

they

are

mostly

apolar

with

and

and

polar

on

the

inner

side

of

the

channel

-LRB-

Fig.

-RRB-

In

electrostatic

terms

the

PfFtn

fourfold

channel

is

similar

to

that

of

both

EcFtnA

and

HuHF

the

outer

entrance

of

it

is

uncharged

as

in

HuHF

and

the

inner

entrance

has

a

compensated

charge

from

the

sequence

of

positive

and

negative

as

in

EcFtnA

The

FC

The

active

site

responsible

for

the

oxidation

of

Fe

-LRB-

II

-RRB-

to

Fe

-LRB-

III

-RRB-

in

ferritins

the

FC

is

located

in

the

central

part

of

each

subunit

-LRB-

Fig.

-RRB-

It

is

generally

believed

that

in

most

ferritins

the

initial

stages

of

the

core

formation

take

place

via

the

FC

Subsequently

iron

incorporation

and

removal

is

thought

to

occur

predominantly

through

one

or

the

other

of

the

channels

formed

around

the

threefold

and

fourfold

symmetry

axes

of

the

In

this

discussion

the

nomenclature

for

the

FCs

in

ferritins

and

BFRs

presented

in

a

recent

review

-LSB-

-RSB-

will

be

followed

The

FC

in

PfFtn

is

of

the

FtnA

-LRB-

EcFtnA

-RRB-

type

which

is

strictly

conserved

in

all

the

closest

prokaryote

structural

homologues

of

PfFtn

besides

EcFtnA

-LRB-

Table

-RRB-

The

EcFtnA

FC

contains

three

metal

binding

sites

-LRB-

A

B

and

C

-RRB-

in

contrast

with

the

HuHF

and

E.

coli

BFR

FC

which

only

contain

two

-LRB-

A

and

B

-RRB-

The

FC

of

the

as-crystallized

PfFtn

showed

iron

bound

to

site

A

only

Iron

binding

to

sites

B

and

C

was

observed

only

after

soaking

the

PfFtn

crystals

in

Fe

-LRB-

II

-RRB-

Table

ferroxidase

center

in

PfFtn

and

its

homologous

crystallized

ferritinsPDB

IDProtein

nameSource

organismResolution

-LRB-

Å

-RRB-

FC

typeMetal

site

occupation

FerritinPyrococcus

FtnAA

-LRB-

Fe

-RRB-

A

B

C

-LRB-

Fe

Zn

-RRB-

FtnAA

B

-LRB-

Zn

-RRB-

aA

B

C

-LRB-

Fe

-RRB-

-LRB-

-RRB-

Thermotoga

FtnAA

-LRB-

Fe

-RRB-

FtnAA

B

-LRB-

Ow

-RRB-

ECFTNAEscherichia

FtnAA

B

-LRB-

Zn

-RRB-

cA

B

C

-LRB-

Fe

-RRB-

ferritinHomo

sapiens

HuHF

ferritin

mutantHomo

sapiens

HuHF

ferritinRana

HuHF

ferritinRana

FC

ferroxidase

centeraZn

in

and

Fe

in

-LSB-

-RSB-

contains

four

water

molecules

two

of

which

are

located

at

the

FC

in

positions

corresponding

to

A

and

B

sites

They

might

be

low-occupancy

metal

sites

but

there

is

no

indication

of

whether

it

is

a

native

or

a

soaked

corresponds

to

the

native

structure

-LSB-

-RSB-

these

sites

were

located

in

the

and

derivatives

respectively

for

which

no

coordinates

were

deposited

in

the

PDB

In

PfFtn

iron

site

A

is

coordinated

by

and

iron

site

B

by

and

and

iron

site

C

by

and

Site

B

is

Å

distant

from

site

A.

Site

C

is

located

and

Å

away

from

sites

A

and

B

respectively

-LRB-

Table

Fig.

-RRB-

and

appear

to

be

monodentate

ligands

therefore

sites

A

and

B

are

coordinated

by

side

chains

from

three

amino

acids

while

site

C

is

coordinated

by

side

chains

from

four

amino

acids

The

final

Fo

Fc

electron

density

around

the

FC

of

a

selected

PfFtn

monomer

in

the

as-isolated

Fe-soaked

and

Zn-soaked

crystal

structures

are

represented

in

Fig.

c

respectively

Table

geometry

statistics

of

the

ferroxidase

center

in

PfFtnBondsaDistance

-LRB-

Å

-RRB-

As-isolatedbFe-soakedbZn-soakedbMA

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MA

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MA

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MA

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MA

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MB

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MB

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MB

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MB

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MC

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MC

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MC

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

MC

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

aM

stands

for

metal

-LRB-

Fe

in

as-isolated

and

in

Fe-soaked

Zn

or

Fe

in

Zn-soaked

-RRB-

bFor

each

column

the

numbers

listed

are

the

mean

value

calculated

from

the

independent

distances

in

the

asymmetric

unit

followed

by

the

corresponding

standard

deviation

in

parentheses

and

finally

the

minimum

and

maximum

values

in

the

population

enclosed

in

bracketsFig

of

the

FC

down

the

PfFtn

subunit

from

the

E-helix

side

The

three

metal

sites

are

annotated

as

A

B

and

C.

Metal

occupation

of

sites

B

and

C

is

observed

only

upon

crystal

soaking

in

Zn

or

Fe

The

dashed

lines

correspond

to

the

coordination

geometries

described

in

Table

The

figure

was

prepared

with

PyMOL

-LSB-

-RSB-

Fig.

of

the

FC

of

a

selected

monomer

in

the

structures

of

as-isolated

Fe-loaded

Fe-soaked

and

Zn-soaked

PfFtn

crystals

a

Final

Fo

Fc

electron-density

map

for

the

as-isolated

PfFtn

contoured

at

map

RMS

b

Final

Fo

Fc

electron-density

map

for

the

Fe-soaked

PfFtn

contoured

at

map

RMS

c

Final

Fo

Fc

electron-density

map

for

the

Zn-soaked

PfFtn

contoured

at

map

RMS

d

Anomalous

Fourier

map

for

the

Fe-loaded

PfFtn

contoured

at

map

RMS

e

Anomalous

Fourier

map

for

the

Fe-soaked

PfFtn

contoured

at

map

RMS

f

Anomalous

Fourier

map

for

the

Zn-soaked

PfFtn

using

the

peak

data

contoured

at

map

RMS

g

Labeled

view

of

the

residues

and

metal

sites

in

the

Fe-soaked

FC

h

Anomalous

Fourier

map

for

the

Zn-soaked

PfFtn

using

the

low-energy

remote

data

contoured

at

map

RMS

i

Dispersive

Fourier

map

for

the

Zn-soaked

PfFtn

contoured

at

map

RMS

The

anomalous

electron-density

maps

were

calculated

using

as

amplitudes

the

anomalous

difference

coefficients

obtained

from

each

dataset

and

the

phases

-LRB-

rotated

by

-RRB-

obtained

from

the

respective

final

structure

refinement

except

for

the

Fe-loaded

data

for

which

the

phases

were

taken

from

the

as-isolated

refinement

The

Zn-dispersive

Fourier

map

was

calculated

by

first

scaling

together

the

peak

and

low-energy

remote

datasets

with

SCALEIT

-LSB-

-RSB-

and

then

using

as

coefficients

the

difference

F

-LRB-

low-energy

remote

-RRB-

F

-LRB-

peak

-RRB-

and

the

phases

from

the

final

Zn-soak

refinement

a

c

show

that

the

occupation

of

site

B

in

the

as-isolated

crystal

is

negligible

d

shows

a

small

residual

occupancy

of

site

B

in

the

Fe-loaded

structure

and

no

evidence

of

site

C

occupancy

e

shows

a

site

B

occupancy

clearly

lower

than

those

of

sites

A

and

C

in

the

Fe-soaked

structure

f

h

and

i

show

that

in

the

Zn-soaked

structure

part

of

the

originally

present

Fe

in

site

A

has

been

displaced

to

site

C

by

the

Zn

ions

that

site

B

is

occupied

by

Zn

only

and

that

site

C

is

very

likely

occupied

by

Fe

only

All

panels

were

drawn

with

the

same

orientation

In

all

panels

except

g

the

bulk

of

PfFtn

monomer

is

represented

as

a

gray

tube

onto

which

the

side

chains

-LRB-

including

Cα

atoms

-RRB-

of

the

FC

residues

have

been

overlaid

in

ball-and-stick

representation

-LRB-

carbon

atoms

blue-gray

oxygen

atoms

red

nitrogen

atoms

blue

iron

atoms

pink

and

zinc

atoms

cyan

-RRB-

In

g

only

the

residues

near

the

FC

are

represented

in

ball-and-stick

mode

and

are

labeled

for

easier

identification

of

the

residues

mentioned

in

the

text

The

figure

was

prepared

with

DINO

-LSB-

-RSB-

For

the

Fe-loaded

crystal

structure

the

anomalous

difference

Fourier

map

calculated

with

the

phases

obtained

from

the

final

as-isolated

refinement

-LRB-

Fig.

-RRB-

shows

a

very

small

occupancy

of

site

B

This

map

is

very

similar

to

that

obtained

from

the

MR

phases

-LRB-

not

shown

-RRB-

In

the

Fe-soaked

crystal

the

anomalous

difference

Fourier

map

calculated

at

the

end

of

the

refinement

showed

that

sites

A

and

C

have

a

clearly

higher

Fe

occupancy

than

site

B

-LRB-

Fig.

-RRB-

In

the

Zn-soaked

crystal

the

anomalous

difference

Fourier

maps

obtained

from

the

two

datasets

collected

-LRB-

just

above

and

eV

below

the

Zn

K-absorption

edge

-RRB-

clearly

show

some

iron

occupation

of

sites

A

and

C

-LRB-

Fig.

-RRB-

whereas

zinc

populates

sites

A

and

B

-LRB-

Fig.

i

-RRB-

The

dispersive

Fourier

map

calculated

using

the

two

datasets

-LRB-

Fig.

-RRB-

shows

Zn

peaks

at

sites

A

and

B

but

since

no

diffraction

data

were

recorded

at

the

inflexion

point

of

the

Zn

K-absorption

edge

this

map

is

noisier

and

no

conclusions

can

be

drawn

about

any

Zn

occupation

of

site

C

Since

the

as-isolated

structure

only

showed

Fe

occupation

of

FC

site

A

we

conclude

that

the

Fe

occupation

of

site

C

in

the

Zn-soaked

structure

results

from

iron

initially

present

in

site

A

displaced

to

site

C

by

the

Zn

soak

This

is

an

interesting

new

result

since

the

previous

Zn-soaked

and

Fe-soaked

structures

reported

for

AfFtn

-LSB-

-RSB-

and

EcFtnA

-LSB-

-RSB-

were

obtained

from

apoferritin

crystals

FC

stability

Interestingly

while

mutagenesis

studies

have

shown

both

sites

A

and

B

to

be

important

for

iron

uptake

and

oxidation

in

ferritins

-LSB-

-RSB-

in

the

crystallized

proteins

the

occupancy

of

the

both

sites

was

found

only

in

BFR

from

D.

desulfuricans

ATCC

-LSB-

-RSB-

The

PfFtn

FC

followed

the

common

trend

and

as

mentioned

already

the

occupation

of

FC

sites

B

and

C

was

only

observed

in

Fe-soaked

or

Zn-soaked

crystals

This

contradicted

our

EPR

studies

that

showed

a

fully

developed

dinuclear

iron

FC

EPR

signal

in

the

sample

prior

to

crystallization

-LSB-

-RSB-

In

order

to

check

the

integrity

of

the

PfFtn

FC

we

loaded

three

samples

of

apo

PfFtn

with

iron

and

incubated

them

for

day

and

months

at

°C

in

air

and

mimicking

the

crystallization

conditions

for

months

in

a

M

ammonium

sulfate

solution

at

room

temperature

The

FC

EPR

signal

from

each

sample

was

measured

after

titration

to

mV

which

is

the

potential

at

which

the

mixed-valence

PfFtn

EPR

signal

is

maximum

-LSB-

-RSB-

The

amplitude

of

the

FC

signal

from

the

sample

incubated

for

months

in

air

was

noticeably

but

not

drastically

smaller

than

that

of

the

sample

incubated

for

day

-LRB-

Fig.

-RRB-

However

the

FC

EPR

signal

amplitude

of

the

sample

incubated

for

months

in

a

M

ammonium

sulfate

solution

was

fivefold

smaller

than

that

from

the

sample

incubated

for

months

in

air

-LRB-

Fig.

-RRB-

The

coordination

distance

from

to

site

B

-LRB-

Table

-RRB-

is

quite

long

-LRB-

more

than

Å

-RRB-

therefore

site

B

may

be

rather

labile

which

could

account

for

its

lower

occupation

in

the

Fe-soaked

and

Zn-soaked

crystals

as

well

as

for

the

easy

removal

of

its

iron

atom

by

the

precipitating

agent

ammonium

sulfate

during

crystallization

of

the

as-isolated

ferritin

Fig.

Fe

-LRB-

III

-RRB-

O

Fe

-LRB-

II

-RRB-

FC

electron

paramagnetic

resonance

-LRB-

EPR

-RRB-

signal

of

μM

PfFtn

titrated

to

mV

The

decrease

in

the

amplitude

of

the

signal

depending

on

incubation

time

and

the

presence

of

crystallization

solution

in

the

sample

illustrates

the

stability

of

the

center

Prior

to

EPR

measurement

the

three

samples

tested

were

incubated

respectively

for

day

after

loading

with

iron

-LRB-

blue

line

-RRB-

for

months

-LRB-

red

line

-RRB-

and

for

months

in

the

presence

of

a

crystallization

solution

M

ammonium

sulfate

-LRB-

black

line

-RRB-

There

is

an

ongoing

discussion

in

the

literature

on

whether

the

FC

iron

sites

A

and

B

are

transient

in

the

process

of

iron

uptake

oxidation

translocation

into

the

protein

core

or

whether

they

form

a

stable

cofactor

-LSB-

-RSB-

In

the

current

experiment

the

decrease

in

the

amplitude

of

the

FC

EPR

signal

in

the

sample

incubated

in

air

for

months

appears

to

be

too

small

to

be

caused

by

iron

translocation

into

the

protein

cavity

Instead

as

shown

by

the

sample

incubated

for

months

in

ammonium

sulfate

the

most

likely

cause

for

the

lack

of

the

B

iron

site

in

the

as-crystallized

PfFtn

is

iron

complexation

and

removal

by

the

crystallization

agent

Thermostability

The

thermal

stability

of

a

protein

can

often

be

described

as

an

equilibrium

between

the

protein

's

folded

and

unfolded

state

and

frequently

correlates

with

the

growth

temperature

of

the

organism

Analysis

of

thermostability

factors

is

often

made

via

comparison

of

the

genome/protein

sequences

from

the

hyperthermophiles

thermophiles

and

mesophiles

or

via

mutagenesis

In

the

literature

a

number

of

factors

have

been

proposed

to

be

involved

in

thermostability

These

include

salt

bridges

hydrogen

bonds

van

der

Waals

interactions

distribution

of

amino

acids

higher

β-strand

content

more

charged

amino

acids

fewer

uncharged

polar

amino

acids

more

hydrophobic

β-branched

amino

acids

and

others

-LSB-

-RSB-

However

no

single

consistent

set

of

determining

factors

has

emerged

from

the

different

studies

that

have

been

carried

out

to

date

therefore

it

appears

that

protein

thermal

stability

is

the

cumulative

result

of

various

factors

Ferritins

are

large

and

highly

symmetrical

agglomerates

of

single

subunits

This

suggests

that

these

proteins

possess

forces

that

keep

the

big

structure

together

Horse

spleen

ferritin

has

been

shown

to

resist

inactivation

by

up

to

M

urea

-LSB-

-RSB-

and

temperatures

up

to

ºC

-LSB-

-RSB-

PfFtn

has

been

shown

to

be

an

extremely

thermostable

protein

No

melting

temperature

could

be

found

with

differential

scanning

calorimetry

up

to

°C

and

the

protein

could

withstand

incubation

at

°C

for

day

or

autoclaving

at

°C

for

half

an

hour

without

loss

of

its

iron-uptake

activity

-LSB-

-RSB-

There

are

no

data

in

the

literature

regarding

the

thermostability

of

other

thermophilic

and

hyperthermophilic

ferritins

In

our

analysis

of

PfFtn

thermostability

-LRB-

summarized

in

Table

-RRB-

and

given

that

no

thermostability

data

are

available

in

the

literature

on

ferritins

from

microorganisms

other

than

P.

furiosus

we

assumed

that

it

is

the

most

thermostable

ferritin

among

those

shown

in

Table

and

we

used

the

optimal

growth

temperature

of

the

organism

as

a

measure

of

relative

thermostability

for

the

other

ferritins

listed

in

Table

However

it

should

be

noted

that

the

denaturation

temperature

of

a

protein

may

be

higher

than

the

optimal

growth

temperature

of

the

organism

where

it

is

produced

Table

bonds

and

salt

bridges

in

the

PfFtn

and

its

closest

structural

homologues

calculated

with

HBPLUSPDB

IDSource

organism

aTotal

H-bonds

bMMMSSSSalt

bridges

cIntramolecularIntermolecularIntramolecularIntermolecularIntramolecularIntermolecularIntramolecularIntermolecular

Pyrococcus

furiosus

-LRB-

°C

-RRB-

vlgThermotoga

maritima

-LRB-

°C

-RRB-

fulgidus

-LRB-

°C

-RRB-

krqCampylobacter

jejuni

-LRB-

°C

-RRB-

eumEscherichia

coli

-LRB-

°C

-RRB-

MM

hydrogen

bonds

between

main-chain

atoms

only

MS

hydrogen

bonds

between

main-chain

and

side-chain

atoms

SS

hydrogen

bonds

between

side-chain

atoms

onlyaOptimal

growth

temperature

in

parenthesesbCalculated

using

default

program

parameterscA

subset

of

SS

hydrogen

bonds

considering

interactions

between

side

chains

of

negatively

charged

Asp/Glu

residues

with

side

chains

of

positively

charged

Arg/His/Lys

residues

The

hydrogen

bonds

were

calculated

with

HBPLUS

-LSB-

-RSB-

using

default

parameters

and

were

divided

into

three

classes

between

main-chain

atoms

only

-LRB-

MM

-RRB-

between

main-chain

and

side-chain

atoms

-LRB-

MS

-RRB-

and

between

side-chain

atoms

only

-LRB-

SS

-RRB-

The

salt

bridges

were

determined

as

a

subset

of

SS

hydrogen

bonds

considering

interactions

between

side

chains

of

negatively

charged

Asp/Glu

residues

with

side

chains

of

positively

charged

Arg/His/Lys

residues

Furthermore

a

distinction

between

intramolecular

-LRB-

within

the

same

monomer

-RRB-

and

intermolecular

-LRB-

between

neighboring

monomers

-RRB-

hydrogen

bonds

was

made

The

MM

bonds

are

mostly

intramolecular

and

are

responsible

for

the

stability

of

the

secondary

structure

elements

-LRB-

α-helices

-RRB-

The

intramolecular

MS

and

SS

hydrogen

bonds

contribute

to

stabilize

the

fold

of

the

monomeric

subunits

The

intermolecular

MS

and

SS

hydrogen

bonds

are

those

that

contribute

most

to

the

stability

of

the

A

surprising

result

of

this

calculation

was

that

the

total

number

of

hydrogen

bonds

in

the

ferritin

was

quite

similar

If

we

exclude

the

lowest

number

-LRB-

for

AfFtn

-RRB-

which

can

possibly

be

attributed

to

its

unusual

quaternary

structure

PfFtn

actually

has

the

lowest

total

number

of

hydrogen

bonds

two

fewer

than

the

ferritin

from

Campylobacter

jejuni

-LRB-

CjFtn

-RRB-

fewer

than

EcFtnA

and

fewer

than

TmFtn

There

is

no

clear

single

trend

in

the

data

listed

in

Table

which

only

emphasizes

the

complex

nature

of

the

structural

factors

governing

thermostability

even

within

a

group

of

closely

related

structures

However

a

careful

inspection

shows

some

interesting

features

which

may

have

a

bearing

on

the

structural

basis

for

the

different

thermostabilities

of

the

different

ferritins

listed

in

Table

The

most

striking

single

feature

in

Table

is

the

marked

increase

in

intramolecular

salt

bridges

between

the

ferritins

of

mesophilic

-LRB-

EcFtnA

and

CjFtn

-RRB-

and

hyperthermophilic

-LRB-

AfFtn

TmFtn

and

PfFtn

-RRB-

organisms

Indeed

a

similar

but

less

clear

trend

is

observed

for

the

SS

hydrogen

bonds

overall

These

structural

features

could

explain

the

higher

resistance

to

thermal

inactivation

in

the

latter

However

this

trend

by

itself

does

not

validate

our

initial

assumption

that

PfFtn

is

the

most

thermostable

ferritin

in

the

group

A

secondary

trend

can

be

perceived

for

the

main-chain

intramolecular

hydrogen

bonds

in

the

hyperthermophilic

organisms

-LRB-

AfFtn

TmFtn

and

PfFtn

-RRB-

which

is

greatest

in

PfFtn

and

may

offset

the

effect

of

a

lower

number

of

intramolecular

salt

bridges

in

comparison

with

TmFtn

Therefore

the

most

important

factor

that

protects

PfFtn

and

other

ferritins

from

thermal

denaturation

appears

to

be

the

preservation

of

the

monomer

fold

rather

than

the

assembly

Conclusions

In

this

study

we

reported

the

crystal

structure

of

PfFtn

High

sequence

similarity

between

PfFtn

TmFtn

and

AfFtn

suggests

a

separate

group

of

thermostable

ferritins

PfFtn

is

the

second

archaeal

ferritin

structure

to

be

reported

following

that

of

AfFtn

In

spite

of

the

high

sequence

identity

-LRB-

-RRB-

between

these

two

proteins

their

quaternary

structures

are

significantly

different

PfFtn

exhibited

the

symmetry

commonly

found

in

other

known

ferritins

whereas

AfFtn

showed

the

symmetry

similar

to

dodecameric

DPS

proteins

The

symmetry

of

the

AfFtn

resulted

in

four

triangular

pores

with

an

approximate

size

of

Å

making

large

openings

in

the

AfFtn

shell

Therefore

the

symmetry

of

the

AfFtn

remains

an

exception

among

ferritins

and

further

research

is

required

to

explain

such

an

unusual

conformation

Three

iron

sites

were

observed

in

the

FC

of

PfFtn

monomer

Site

A

was

present

in

the

as-crystallized

protein

and

sites

B

and

C

were

observed

only

upon

crystal

soaking

with

either

Fe

or

Zn

ions

Combining

these

results

with

our

measurements

of

the

FC

EPR

signal

prior

to

crystallization

where

samples

were

incubated

in

air

for

day

and

months

and

for

months

in

M

ammonium

sulfate

-LRB-

the

crystallization

solution

-RRB-

we

conclude

that

the

lack

of

iron

atoms

at

the

B

site

is

very

likely

due

to

their

complexation

by

ammonium

sulfate

and

that

the

absence

of

these

sites

in

the

as-crystallized

protein

can

not

be

taken

per

se

as

evidence

of

a

transient

nature

of

the

FC

in

PfFtn

and

other

ferritins

The

possible

iron

entry

and

exit

routes

of

PfFtn

the

threefold

and

fourfold

channels

are

similar

to

those

from

bacterial

ferritins

in

the

sense

that

the

threefold

channel

is

less

hydrophilic

and

the

fourfold

channel

is

more

polar

than

the

channels

of

mammalian

H

and

L

chain

ferritins

The

hyperthermostability

of

PfFtn

was

analyzed

at

the

structural

level

by

comparing

the

number

of

salt

bridges

and

hydrogen

bonds

in

PfFtn

with

those

in

AfFtn

TmFtn

CjFtn

and

EcFtnA

Although

there

are

no

data

on

the

thermal

stability

of

other

thermostable

ferritins

our

results

suggest

that

TmFtn

the

ferritin

from

T.

maritima

a

bacterium

which

grows

optimally

at

°C

is

probably

more

thermostable

than

PfFtn

Owing

to

a

high

number

of

intramolecular

hydrogen

bonds

between

main-chain

atoms

and

between

main-chain

and

side-chain

atoms

the

main

factor

contributing

to

the

high

thermostability

of

PfFtn

as

well

as

the

other

structurally

similar

ferritins

from

hyperthermophilic

microorganisms

appears

to

be

the

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Neck pain in chronic whiplash syndrome treated with botulinum toxin . A double-blind , placebo-controlled clinical trial Objectives Neck pain in chronic whiplash syndrome is a major burden for patients , healthcare providers and insurance companies . Randomized data on treatment of botulinum toxin in chronic whiplash syndrome are scarce . We conducted a randomized , placebo-controlled clinical trial to prove efficacy of botulinum toxin for neck pain in chronic whiplash syndrome . Introduction Intractable chronic neck pain after a whiplash trauma is a major burden for patients , healthcare providers and insurance companies . It is estimated that 14 -- 42 % of patients develop chronic symptoms after an acute whiplash trauma -LSB- 2 -RSB- . These patients often have reduced mobility of the neck with tender muscles and increased muscle activity -LSB- 25 -RSB- . Pathophysiology is still unknown , but myofascial triggerpoints can develop in cervical muscles from excessive neck and shoulder muscle contraction due to an overt injury such as acceleration of the neck in a motor vehicle accident -LSB- 5 -RSB- . Botulinum toxin is used to treat a variety of neurological disorders associated with pathologically increased muscle tone . Two previous studies have reported that botulinum toxin might be successful in patients with chronic neck pain following whiplash injuries -LSB- 6 , 12 -RSB- . These studies were uncontrolled and the follow-up period was relatively short . In a randomized placebo - controlled trial involving 26 patients with chronic neck complaints after whiplash injury a significant improvement on total pain was suggested -LSB- 7 , 8 -RSB- . This study however has some methodological problems , such as a relatively short follow-up period of 4 weeks and a small sample size -LSB- 7 , 8 -RSB- . Based upon the present evidence botulinum toxin can not be recommended as standard treatment for chronic neck pain after whiplash injury . We therefore conducted a double blind placebo-controlled randomized trial to confirm if botulinum toxin in treatment of neck pain is indeed effective in chronic whiplash syndrome . Methods Patients Patients were eligible if they had a whiplash type neck distortion defined as a soft tissue injury of the neck following a vehicle collision , with symptoms lasting longer than 6 months . They also met criteria of the Quebec Task Force grade 1 or 2 whiplash-associated disorders -LRB- WAD -RRB- . WAD grade 1 means only neck pain and stiffness and grade 2 requires also musculoskeletal signs , e.g. a decreased range of motion -LSB- 24 -RSB- . Exclusion criteria were : age < 18 ; pregnancy ; neuromuscular disorders ; previous use of other investigational new drugs in the past 30 days prior to the screening visit ; previous use of botulinum toxin ; traumatic abnormalities on radiological examinations . The institutional ethics committee approved the study protocol , and patients gave informed consent before inclusion . Study design The trial had a double-blind , placebo-controlled parallel design . After patient 's consent was obtained , we recorded demographics -LRB- age , gender -RRB- . During a baseline-period of 4 weeks , patients used a diary to record the presence of neck pain , number of neck pain hours per day , number of days on which medication was taken , and number of tablets taken per day . At the end of this baseline-period -LRB- week 0 -RRB- patients recorded their mean neck pain intensity scored on 100 mm Visual Analogue Scale of the previous 4 weeks -LRB- week -- 4 to 0 , VAS-pre-treatment -RRB- . At week 0 , patients were randomly assigned to receive 100 units botulinum toxin -LRB- Botox ® -RRB- or placebo -LRB- saline -RRB- in 2 cc syringes . At 12 weeks follow-up they recorded again their mean neck pain intensity of the previous 12 weeks -LRB- week 0 to 12 , VAS-post-treatment -RRB- . The local trial pharmacist prepared the drug , coded the syringes , and kept treatment codes . The pharmacist had no further participation in the trial . An experienced clinical neurophysiologist -LRB- D.T. -RRB- performed all injections . Injection sites were selected individually -LRB- i.e. ` follow the pain ' approach -RRB- in muscles with clinically increased muscle tone or muscle tenderness , and included m. occipitofrontalis , m.temporalis , m. masseter , m. sternocleidomastoideus , m. splenius capitis , m. trapezius and m. semispinalis . Outcomes After patients were injected at week 0 in one single treatment-session , clinical characteristics were recorded using diaries during the 12 weeks post-treatment . The primary outcome measure was the intensity of the neck pain of the total study period , week 0 -- 12 , scored on the VAS at week 12 -LRB- one single measurement -RRB- compared with baseline -LRB- VAS week -- 4 to 0 -RRB- . Patients were asked to rate -LRB- in a single value -RRB- severity of neck pain which at times may be mild and perhaps at others severe , by `` integrating severity over time '' . Secondary outcome measures were average number of neck pain days , average number of neck pain hours per day , average number of days on which symptomatic treatment was taken , mean number of symptomatic tablets per day at 12 weeks compared with baseline . Additionally , a blinded investigator asked patients whether there was any improvement in their condition after 4 , 8 and 12 weeks in the two treatment groups -LRB- five-point scale : great worsening , any worsening , no improvement , any improvement and great improvement -RRB- . Range of cervical motion was measured with a cervical range of motion -LRB- CROM -RRB- device to measure the six conventional movements of the spine at week 12 compared with baseline . For measurements of CROM , the patients were seated in a chair with their shoulders supported on a backrest . Patients were instructed to assume a comfortable position and then to perform each required movement and return to the start position . The movements were assessed in this order : right rotation , left rotation , right flexion , left flexion , extension , flexion and then summated to determine the mean total range of motion . We also divided all patients in responders and non-responders at week 12 . We considered at least 45 % reduction in the VAS compared with baseline to be clinically relevant -LRB- e.g. responder -RRB- . All adverse events during the 3 months follow-up were recorded by the investigators . Sample size determination At baseline we expected mean neck pain intensity on the VAS of 40/100 mm . The estimated placebo effect was a 8 mm reduction to a VAS intensity of 32/100 at 12 weeks . We hypothesized a 18 mm reduction in the treatment group to a VAS intensity of 22/100 , although there are no earlier data on treatment effect of BTX in these patients . To detect this clinically relevant effect -LRB- 8 versus 18 mm improvement -RRB- at a 5 % level of statistical significance -LRB- two-tailed -RRB- , with a power of 80 % , a total of 40 patients was needed . Statistical analysis We calculated mean differences between baseline VAS data and VAS data after 12 weeks , using the t-test ; statistical uncertainty was expressed in 95 % confidence intervals . The responders at week 12 in each treatment group and the differences in improvement scores were compared by use of the Chi-square test . All these analysis were done with SPSS -LRB- version 10.0 -RRB- . Results Between November 1999 and March 2003 40 patients were included at Leyenburg hospital The Hague . Of the 59 patients assessed for eligibility , 19 patients were excluded . Four patients did not meet the inclusion criteria , eight patients refused to participate and seven patients withdrew in the baseline period . No patient was lost to follow-up . In 1 patient the neck pain diary was incomplete during the whole study period -LRB- missing data on neck pain duration , days on which medication was taken and mean number of tablets per day -RRB- . In 7 patients the neck pain diary for the post-treatment period was partly incomplete -LRB- missing data on neck pain duration , days on which medication was taken and mean number of tablets per day -RRB- . There were no differences in baseline characteristics between the two treatment groups . Neck pain intensity , VAS at baseline was 64.5 mm -LRB- SD = 14.8 -RRB- in the botulinum group and 62.1 -LRB- SD = 20.3 -RRB- in the placebo group -LRB- Table 1 -RRB- . Table 1Baseline characteristicsCharacteristicBotulinum toxin -LRB- n = 20 -RRB- Placebo -LRB- n = 20 -RRB- Mean age , years -LRB- range -RRB- 39 -LRB- 20 -- 58 -RRB- 34 -LRB- 20 -- 49 -RRB- Female sex -LRB- % -RRB- 14 -LRB- 70 % -RRB- 13 -LRB- 65 % -RRB- Ongoing claims15 -LRB- 75 % -RRB- 18 -LRB- 90 % -RRB- Mean duration of pain , months -LRB- SD -RRB- 40 -LRB- 71 -RRB- 37 -LRB- 20 -RRB- Neck pain severity , mean VAS -LRB- SD -RRB- 64.5 -LRB- 14.8 -RRB- 62.1 -LRB- 20.3 -RRB- Mean number of neck pain days , % -LRB- SD -RRB- 97 % -LRB- 11 -RRB- * 196 % -LRB- 12 -RRB- Mean neck pain duration per day , hours -LRB- SD -RRB- 12.7 -LRB- 4.0 -RRB- * 311.7 -LRB- 4.5 -RRB- Mean number of medication days , % -LRB- SD -RRB- 50 % -LRB- 41 -RRB- * 136 % -LRB- 40 -RRB- Mean number of analgesics per day , tablets -LRB- SD -RRB- 1.4 -LRB- 1.9 -RRB- 1.3 -LRB- 2.1 -RRB- Mean total cervical range of motion , degrees -LRB- SD -RRB- 266 -LRB- 73 -RRB- * 1266 -LRB- 60 -RRB- * 1 * 1 = one missing data ; * 3 = three missing data At 12 weeks the VAS scores were 52.0 mm -LRB- SD = 29.2 -RRB- in the botulinum group and 56.7 mm -LRB- SD = 29.6 -RRB- in the placebo group . Mean differences between VAS pre - and post-treatment was 12.5 in the botulinum group and 5.5 in the placebo group . The therapeutic gain -LRB- = difference in improvement between the two treatment groups measured in mm -RRB- was -- 7.0 -LRB- p = 0.31 ; 95 % CI -LRB- -- 20.7 to + 6.7 -RRB- -RRB- . Six patients in the botulinum group were responders as defined , versus four in the placebo group -LRB- NS , p = 0.5 -RRB- . Patients ' assessment of improvement/worsening showed no significant difference between the two groups at week 4 -LRB- p = 0.9 -RRB- , week 8 -LRB- p = 0.4 -RRB- and 12 -LRB- p = 0.4 -RRB- -LRB- Table 2 -RRB- . Table 2Improvement scaleImprovementAt 4 weeksAt 8 weeksAt 12 weeksBotulinum toxin -LRB- n = 20 -RRB- Placebo -LRB- n = 20 -RRB- Botulinum toxin -LRB- n = 20 -RRB- Placebo -LRB- n = 20 -RRB- Botulinum toxin -LRB- n = 20 -RRB- Placebo -LRB- n = 20 -RRB- Great worsening400212Any worsening241423No improvement688768Any improvement555454Great improvement236363Total19 * 12020202020 * 1 = one missing data There were no significant differences between the two treatment groups in mean number of neck pain days , mean number of neck pain hours per day , mean number of days on which medication was taken , and mean number of tablets taken per day pre - and post-treatment -LRB- Table 3 -RRB- . Table 3Secondary outcome measuresOutcomeBotulinum toxin -LRB- n = 20 -RRB- Placebo -LRB- n = 20 -RRB- Therapeutic gain -LRB- 95 % CI -RRB- Neck pain days -LRB- % ± SD -RRB- Baseline -LRB- 4 weeks -RRB- 97 % -LRB- 11 -RRB- * 196 % -LRB- 12 -RRB- Post-treatment -LRB- 12 weeks -RRB- 89 % -LRB- 23 -RRB- * 289 % -LRB- 25 -RRB- * 2 Mean difference8 % -LRB- 18 -RRB- 7 % -LRB- 22 -RRB- − 1 % -LRB- − 15 % to + 13 % -RRB- Neck pain duration -LRB- hours/day ± SD -RRB- Baseline -LRB- 4 weeks -RRB- 12.7 -LRB- 4.0 -RRB- * 311.7 -LRB- 4.5 -RRB- Post-treatment -LRB- 12 weeks -RRB- 12.2 -LRB- 4.6 -RRB- * 711.0 -LRB- 5.1 -RRB- * 2 Mean difference0 .86 -LRB- 2.2 -RRB- 0.72 -LRB- 4.7 -RRB- − 0.14 -LRB- − 3.0 to + 2.7 -RRB- Days on which analgesics were taken -LRB- % ± SD -RRB- Baseline -LRB- 4 weeks -RRB- 49 % -LRB- 42 % -RRB- 35 % -LRB- 40 % -RRB- Post-treatment -LRB- 12 weeks -RRB- 42 % -LRB- 38 % -RRB- * 324 % -LRB- 32 % -RRB- * 2 Mean difference6 .4 % -LRB- 16 % -RRB- 5.7 % -LRB- 24 % -RRB- 0.7 % -LRB- − 15 % to + 13 % -RRB- Number of analgesics taken per day -LRB- tablets/day ± SD -RRB- Baseline -LRB- 4 weeks -RRB- 1.4 -LRB- 1.9 -RRB- * 11.3 -LRB- 2.1 -RRB- Post-treatment -LRB- 12 weeks -RRB- 1.2 -LRB- 1.8 -RRB- * 20.95 -LRB- 1.9 -RRB- * 1 Mean difference0 .32 -LRB- 0.8 -RRB- 0.18 -LRB- 0.6 -RRB- − 0.14 -LRB- − 0.6 to + 0.4 -RRB- Total cervical range of motion -LRB- degrees ± SD -RRB- Baseline266 -LRB- 73 -RRB- * 1266 -LRB- 60 -RRB- * 1 Post-treatment -LRB- week 12 -RRB- 293 -LRB- 59 -RRB- * 2277 -LRB- 62 -RRB- * 3 Mean difference22 -LRB- 43 -RRB- 11 -LRB- 36 -RRB- − 11 -LRB- − 40 to + 17 -RRB- * 1 = one missing data ; * 2 = two missing data ; * 3 = three missing data * 7 = seven missing data At baseline all patients showed a reduced cervical range of motion -LRB- CROM -RRB- in all directions -LSB- 9 -RSB- . There was a slight improvement in total CROM in favour of the botulinum group . However , CROM was not significantly different between BTX group and placebo , nor between responders and non-responders . Twenty-seven patients reported minor side-effects , 15 in the botulinum toxin group and 12 in the placebo group . The main complaint was `` short-lasting pain at the injection sites '' . Discussion Our study could not demonstrate botulinum toxin to be more effective than placebo in treatment of neck pain in patients with chronic whiplash syndrome . Previous open label studies with botulinum toxin in patients with chronic whiplash syndrome suggested some beneficial effect on neck pain -LSB- 12 -RSB- . Our study is the second published randomized-controlled trial to determine efficacy of botulinum toxin in patients with chronic whiplash syndrome . The other randomized placebo-controlled trial in 26 patients with chronic whiplash syndrome reported a beneficial effect of botulinum toxin -LSB- 7 , 8 -RSB- . This study , however , has some methodological shortcomings . Primary outcome was improvement of headache intensity scored on the VAS after 4 weeks compared with baseline . The post treatment difference between the two treatment groups was reported to be significant . However , baseline-characteristics were significantly different , with a significant higher headache intensity in the botulinum group at baseline . -LRB- VAS 6.5 -LRB- range 2 -- 9 -RRB- versus 3.0 -LRB- 0 -- 8 -RRB- in the placebo group -LRB- p < 0.01 -RRB- -RRB- . The reported improvement may mainly have been caused by the unequal distribution of the patients at baseline . In their second publication -LRB- on the same trial -RRB- the mean total pain score was reported , ranging from 0 -- 30 , expressing the total pain experience for neck pain , headache and shoulder pain . After 4 weeks the mean total pain was 10.0 -LRB- SE 1.3 -RRB- in the botulinum group and 14.1 -LRB- SE 21 -RRB- in the placebo group , which was also reported to be significant . These data are difficult to interpret without individual scores . Calculation of the standard deviation could suggest that some patients even had a negative mean total pain score . In any case , the distribution was truncated and skew to the right . There are some points of discussion in our study . We used injections at trigger points on an individual basis , instead of following a standardized protocol . This might seem subjective and arbitrary , but resembles clinical practice and is usually recommended for botulinum toxin -LRB- BTX -RRB- treatment in pain syndromes -LSB- 10 , 16 -RSB- . We used doses of 100 U Botox ® , which should be sufficient for a single treatment session . There was only slight , not significant , improvement of cervical range of motion in the botulinum group , suggesting some , if any , effect on muscle tenderness . Also , none of our patients reported muscular weakness as a side effect . Therefore , further increase of the dosage of botulinum toxin might be possible . There is some evidence that multiple treatment sessions are needed for sufficient clinical benefit -LSB- 20 -RSB- . In our study there was not even a trend for a positive treatment effect after this single treatment session of BTX . Still , a positive effect of multiple treatment injections can not be entirely ruled out -LSB- 16 -RSB- . Pathophysiology of chronic pain in whiplash syndrome is still a matter of debate . There is evidence that myofascial pain is a major factor in chronic pain syndromes like chronic neck pain and chronic tension-type headache -LSB- 14 -RSB- . The hypothesis of BTX being effective in chronic whiplash syndrome could be pain reduction by decreasing muscle tenderness and/or activity . If increased muscle tenderness is a key feature in chronic neck pain after a whiplash injury , a more beneficial effect of BTX would have been expected . However , in other chronic pain syndromes where muscle tenderness is thought to play a role BTX was also proven ineffective -LSB- 17 , 23 -RSB- . BTX might only be effective in neck pain in combination with cervical dystonia -LSB- 26 -RSB- . In our study were no patients with cervical dystonia . Patients with chronic whiplash syndrome also form a very heterogeneous group , with a wide spectrum of symptoms , varying from neck pain to headache , with pain of different degrees . Many different factors seem to play a role in the development of acute and chronic pain after a whiplash injury . There is ample evidence that central mechanisms might play a more important role in the development of chronic neck pain in whiplash patients -LSB- 4 -RSB- , as in other chronic pain syndromes . Damaged peripheral tissue releases inflammatory mediators -LRB- like bradykinine and prostaglandins -RRB- that changes the sensitivity of high-threshold nociceptors -LRB- peripheral sensitization -RRB- . This , in turn , sets up changes in the responsiveness of neurons in the central nervous system -LRB- CNS -RRB- , ultimately increasing the excitability of neurons within the CNS , representing central sensitization -LSB- 21 -RSB- . Patients with chronic whiplash syndrome display exaggerated pain after sensory stimulation as a consequence of this state of hyperexcitability of the central nociceptive system -LSB- 1 , 13 -RSB- . In other words , an alteration in function of the central nervous system , the way it responds to normal inputs , seems present in these patients . This central hypersensivity is probably independent of the nociceptive input arising from the painful and tender muscles -LSB- 4 -RSB- , and possibly a consequence of deficient pain inhibitory systems . Many somatic , psychological or cultural factors may influence central sensitization . Different studies show that prognosis of chronic whiplash syndrome is also dependent on cultural factors -LSB- 15 , 18 , 22 -RSB- . It has even been shown that presence or absence of compensation claims is one of the most important predictors of bad outcome in patients after whiplash injury -LRB- also present in our group in 75 -- 90 % of the patients -RRB- -LSB- 11 , 19 -RSB- . The etiological contribution of all different psychological factors , like for example coping style -LSB- 3 -RSB- , to central sensitization is not yet clarified . It is not to be predicted that BTX will improve all different factors , especially psychological and cultural factors , which may contribute to development of this chronic whiplash syndrome . Based on present evidence BTX can not be recommended as treatment for neck pain in chronic whiplash patients . Future studies directed on possible central mechanisms of this complicated chronic pain syndrome are warranted . Contributors The trial was designed by S.F.T.M. de Bruijn , D.L.J. Tavy and M. Padberg . Statistical analysis was performed by M. Padberg . Conflict of interest statement Non declared .

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O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O B I I O O O O O O B I O O O B I I O O O O O B I O O O O O O O O O O O O O O O O B I O O O B I I O O O O O O O O O O B I I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

neck pain
chronic whiplash syndrome
botulinum toxin