drug

resistance

genotyping

from

dried

blood

spots

stored

for

year

at

°C

Background

Dried

blood

spots

-LRB-

DBSs

-RRB-

are

an

attractive

alternative

to

plasma

for

drug

resistance

testing

in

resource-limited

settings

We

recently

showed

that

can

be

efficiently

genotyped

from

DBSs

stored

at

°C

for

prolonged

periods

-LRB-

years

-RRB-

Here

we

evaluated

the

efficiency

of

genotyping

from

DBSs

stored

at

°C

for

year

Introduction

The

availability

of

clinical

specimens

that

can

be

easily

collected

stored

and

transported

is

advantageous

in

areas

that

lack

appropriate

infrastructure

for

blood

processing

Whole

blood

samples

collected

by

finger

stick

and

dried

onto

a

filter

paper

-LSB-

dried

blood

spots

-LRB-

DBSs

-RRB-

-RSB-

represent

an

attractive

alternative

to

the

conventional

collection

of

blood

in

tubes

DBSs

have

been

extensively

used

for

antibody

testing

molecular

and

virus

load

-LRB-

VL

-RRB-

and

are

now

considered

a

convenient

alternative

to

plasma

for

drug

resistance

testing

We

recently

found

that

drug

resistance

genotypes

generated

from

DBSs

were

similar

to

those

derived

from

plasma

in

antiretroviral-naive

and

experienced

patients

We

also

found

that

resistance

testing

from

DBSs

can

be

as

sensitive

as

with

plasma

when

using

a

genotypic

assay

that

amplifies

a

large

-LRB-

kb

-RRB-

pol

fragment

The

high

success

of

amplification

was

noted

using

DBSs

stored

at

°C

in

the

presence

of

desiccant

suggesting

that

°C

may

represent

a

feasible

storage

temperature

for

DBSs

Although

these

findings

were

encouraging

storage

at

°C

is

not

always

possible

in

resource-limited

settings

where

°C

or

room

temperature

may

represent

a

more

realistic

alternative

Studies

on

VL

determinations

using

DBSs

have

reported

the

efficient

amplification

of

short

RNA

sequences

from

filter

papers

stored

at

°C

or

room

temperature

However

genotypic

assays

usually

rely

on

the

amplification

of

large

pol

fragments

and

are

thought

to

be

particularly

sensitive

to

RNA

degradation

that

may

occur

under

suboptimal

storage

conditions

Here

we

report

on

the

efficiency

of

amplification

and

drug

resistance

genotyping

from

DBSs

stored

at

°C

for

year

Methods

Preparation

and

storage

of

DBSs

We

prepared

a

total

of

DBSs

from

residual

diagnostic

specimens

collected

from

highly

antiretroviral-experienced

subtype

B-infected

persons

A

more

detailed

description

of

the

study

population

including

antiretroviral

drug

treatment

has

been

reported

elsewhere

Drug

resistance

genotypes

were

available

for

the

matched

plasma

specimens

The

median

plasma

VL

in

these

patients

was

RNA

copies/mL

-LRB-

range

Versant

RNA

Assay

Bayer

HealthCare

Diagnostic

Division

Tarrytown

NY

USA

-RRB-

-LRB-

Table

-RRB-

DBSs

were

prepared

by

pipetting

µL

of

whole

blood

onto

pre-marked

circles

on

filter

paper

cards

-LRB-

Schleicher

Schuell

Keene

NH

USA

-RRB-

Cards

were

dried

overnight

at

room

temperature

-LRB-

°C

-RRB-

placed

in

a

gas-impermeable

sealable

plastic

bag

-LRB-

Fisher

Scientific

Company

Pittsburgh

PA

USA

-RRB-

containing

a

silica

gel

desiccant

-LRB-

Mini

Pax

Sorbent

Multisorb

technologies

Buffalo

NY

USA

-RRB-

and

stored

at

°C

until

they

were

shipped

to

CDC

in

dry

ice

Upon

arrival

individual

plastic

bags

were

put

in

a

larger

bag

containing

three

extra

desiccants

and

stored

at

°C

All

desiccants

were

evaluated

for

humidity

at

months

of

the

bags

had

evidence

of

humidity

as

indicated

by

the

colour

indicator

in

the

desiccant

Therefore

a

new

desiccant

was

added

to

each

individual

bag

after

allowing

the

spots

to

equilibrate

at

room

temperature

for

min

of

the

specimens

had

mould

after

months

of

storage

at

°C

Table

Frequency

of

amplification

of

pol

from

plasma

or

DBSs

stored

at

°C

for

months

or

at

°C

for

months

Specimen

ID

Plasma

VL

-LRB-

copies/mL

-RRB-

Plasmaa

DBS

-LRB-

°C

months

ViroSeq

assay

-RRB-

b

DBS

-LRB-

°C

months

ViroSeq

assay

-RRB-

DBS

-LRB-

°C

months

in-house

assay

-RRB-

n.s

n.s

n.s.

n.s.

n.s.

n.s.

n.s.

RT

and

protease

were

amplified

but

failed

to

generate

full-length

sequences

aPlasma

genotypes

were

generated

using

the

Viroseq

assay

except

for

and

-LRB-

TrueGene

assay

-RRB-

bAmplification

from

these

DBSs

stored

at

°C

has

been

reported

elsewhere

Nucleic

acid

extraction

Total

nucleic

acids

were

extracted

from

one

spot

after

year

of

storage

at

°C

using

a

modification

of

the

Nuclisens

assay

described

previously

Briefly

a

whole

spot

containing

µL

of

blood

was

cut

with

scissors

and

added

into

mL

of

Nuclisens

lysis

buffer

-LRB-

bioMérieux

Inc.

Durham

NC

USA

-RRB-

Special

care

was

taken

to

avoid

contact

of

the

scissors

with

the

blood

spots

scissors

and

forceps

were

sprayed

with

ethanol

after

each

use

and

wiped

dry

After

h

of

incubation

with

lysis

buffer

at

room

temperature

under

gentle

rotation

the

supernatant

was

clarified

by

centrifugation

at

g

for

min

and

then

transferred

to

a

clean

mL

conical

tube

Nucleic

acids

were

then

extracted

following

the

manufacturer

's

instructions

resuspended

in

µL

of

elution

buffer

and

stored

at

°C

until

use

Drug

resistance

testing

Resistance

testing

from

DBSs

was

performed

using

the

ViroSeq

Genotyping

System

-LRB-

Abbott

Molecular

Des

Plains

IL

USA

-RRB-

and

an

in-house

reverse

transcriptase

-LRB-

RT

-RRB-

nested

PCR

method

described

previously

The

ViroSeq

assay

amplifies

a

kb

pol

fragment

and

has

a

sensitivity

of

detection

of

RNA

copies/mL

plasma

The

in-house

assay

amplifies

a

bp

fragment

of

pol

comprising

amino

acids

of

the

protease

and

of

the

RT.

This

assay

has

been

validated

only

for

subtype

B

viruses

and

has

a

sensitivity

of

detection

of

RNA

copies/mL

of

plasma

Genotypes

were

interpreted

using

the

Stanford

Genotypic

Resistance

Interpretation

Algorithm

-LRB-

version

-RRB-

available

at

Results

When

the

ViroSeq

assay

was

used

only

of

the

-LRB-

-RRB-

DBS

specimens

stored

at

°C

were

successfully

genotyped

of

the

successful

amplifications

were

from

specimens

that

had

plasma

viraemia

RNA

copies/mL

-LRB-

Table

-RRB-

An

additional

five

specimens

produced

amplicons

that

did

not

generate

full-length

RT

and

protease

sequences

Visual

inspection

of

the

desiccant

bags

after

months

of

storage

at

°C

showed

evidence

of

humidity

in

of

the

specimens

Of

these

specimens

only

those

with

plasma

VL

higher

than

RNA

copies/mL

-LRB-

specimens

-RRB-

were

successfully

genotyped

by

the

ViroSeq

assay

at

months

-LRB-

data

not

shown

-RRB-

As

we

previously

noted

using

parallel

DBSs

stored

at

°C

resistance

genotypes

generated

from

DBSs

and

matched

plasma

specimens

were

highly

concordant

Of

the

drug

resistance

mutations

identified

in

these

plasma

sequences

were

also

found

in

sequences

generated

from

DBSs

Of

the

five

mutations

absent

in

DBSs

three

were

protease

mutations

two

were

major

-LRB-

and

-RRB-

and

one

minor

-LRB-

-RRB-

and

two

were

polymorphisms

at

codon

of

the

RT

-LRB-

and

-RRB-

-LRB-

data

not

shown

-RRB-

We

also

compared

the

efficiency

of

amplification

seen

in

these

DBSs

with

that

previously

seen

in

DBSs

prepared

in

parallel

from

the

same

patients

and

stored

at

°C

for

months

Under

storage

at

°C

all

of

the

DBS

specimens

were

successfully

amplified

and

genotyped

-LRB-

Table

-RRB-

The

ViroSeq

assay

amplifies

a

large

-LRB-

kb

-RRB-

pol

fragment

in

a

single

round

of

PCR

and

may

be

particularly

sensitive

to

the

degradation

of

nucleic

acids

that

may

occur

during

long-term

storage

We

therefore

assessed

if

the

rate

of

amplification

from

DBSs

stored

at

°C

could

be

improved

by

using

an

in-house

assay

that

amplifies

a

smaller

-LRB-

bp

-RRB-

fragment

and

uses

a

nested

PCR

step

Using

this

method

viruses

from

of

the

DBSs

-LRB-

-RRB-

were

successfully

genotyped

-LRB-

Table

-RRB-

Despite

the

use

of

a

different

assay

to

genotype

viruses

from

these

DBS

specimens

resistance

genotypes

were

highly

concordant

with

those

generated

from

plasma

using

the

ViroSeq

assay

of

the

mutations

found

in

plasma

viruses

were

also

found

in

viruses

from

DBSs

-LRB-

data

not

shown

-RRB-

To

investigate

if

the

decreased

amplification

success

rate

observed

with

the

ViroSeq

assay

was

due

to

insufficient

or

degraded

HIV

nucleic

acids

we

performed

serial

dilutions

of

RNA

extracts

from

high

VL

specimens

and

tested

them

by

both

the

in-house

and

the

ViroSeq

assays

-LRB-

Figure

-RRB-

Although

both

assays

have

comparable

sensitivity

amplification

signals

were

only

maintained

when

the

shorter

pol

fragment

was

amplified

by

the

in-house

method

Figure

shows

that

such

signals

were

consistently

lost

when

the

larger

pol

fragment

was

amplified

using

the

ViroSeq

assay

suggesting

the

possibility

of

nucleic

acid

degradation

during

storage

at

°C

Such

a

possibility

was

further

supported

by

parallel

testing

of

serial

dilutions

of

RNA

extracts

prepared

from

specimens

stored

at

°C

Figure

-LRB-

c

-RRB-

shows

that

amplification

signals

generated

from

these

specimens

using

the

in-house

assay

were

generally

stronger

than

those

obtained

from

the

same

DBS

specimens

stored

at

°C

-LRB-

Figure

-RRB-

Figure

Agarose

gel

showing

the

pol

amplification

signals

obtained

from

DBS

specimens

stored

at

or

°C

Two

serial

dilutions

-LRB-

and

-RRB-

of

specimens

-LRB-

RNA

copies/mL

-RRB-

-LRB-

RNA

copies/mL

-RRB-

-LRB-

RNA

copies/mL

-RRB-

and

-LRB-

RNA

copies/mL

-RRB-

were

prepared

in

Nuclisens

elution

buffer

and

were

tested

in

parallel

using

the

ViroSeq

assay

-LRB-

kb

-RRB-

or

the

in-house

RT-nested

PCR

method

-LRB-

bp

-RRB-

-LRB-

a

-RRB-

DBS

specimens

stored

at

°C

and

amplified

using

the

ViroSeq

assay

-LRB-

b

-RRB-

DBS

specimens

stored

at

°C

and

amplified

using

the

in-house

method

-LRB-

c

-RRB-

DBS

specimens

stored

at

°C

and

amplified

using

the

in-house

method

Discussion

Our

study

on

a

small

number

of

specimens

suggests

that

storage

at

°C

may

represent

a

feasible

alternative

to

°C

for

long-term

storage

of

DBSs

However

we

noted

that

the

efficient

genotyping

from

these

DBS

specimens

required

the

use

of

an

in-house

nested

PCR

assay

and

that

the

ViroSeq

assay

failed

to

genotype

a

substantial

proportion

of

specimens

The

results

with

the

ViroSeq

assay

differed

from

our

previous

findings

with

this

assay

showing

a

high

success

of

genotyping

from

DBSs

stored

at

°C

and

suggest

that

some

degradation

of

RNA

may

have

occurred

during

long-term

storage

at

°C

possibly

due

to

suboptimal

storage

temperature

humidity

or

both

Such

a

possibility

was

suggested

by

the

weaker

amplification

signals

observed

in

RNA

extracts

from

specimens

stored

at

°C

compared

with

those

stored

at

°C

upon

serial

dilutions

Although

different

efficiencies

of

RNA

extraction

could

partially

explain

these

differences

we

found

that

a

high

proportion

of

the

specimens

stored

at

°C

had

evidence

of

humidity

at

months

whereas

of

the

specimens

stored

at

°C

did

High

humidity

conditions

are

thought

to

be

detrimental

to

resistance

testing

from

DBSs

given

the

extreme

sensitivity

of

HIV

nucleic

acids

to

degradation

in

the

presence

of

humidity

Nonetheless

we

showed

that

it

is

possible

to

overcome

potential

losses

in

RNA

integrity

and

efficiently

genotype

from

DBSs

stored

at

°C

by

using

an

in-house

nested

PCR

protocol

that

amplifies

a

smaller

fragment

using

stringent

quality-controlled

reagents

These

results

are

encouraging

and

expand

on

our

earlier

findings

showing

the

efficient

genotyping

of

pol

from

DBSs

stored

at

°C

for

up

to

years

A

high

success

of

genotyping

has

also

been

noted

from

DBSs

stored

for

months

at

°C

and

humidity

in

the

presence

of

desiccant

Under

these

extreme

conditions

viruses

from

of

the

DBS

specimens

could

be

genotyped

by

using

in-house

nested

PCR

assays

that

amplified

small

-LRB-

bp

-RRB-

overlapping

pol

sequences

The

use

of

nested

PCR

protocols

and/or

the

amplification

of

small

fragments

may

likely

result

in

higher

amplification

success

rates

from

DBSs

stored

under

less

optimal

conditions

Such

a

possibility

was

supported

by

our

findings

showing

a

better

maintenance

of

amplification

efficiencies

using

the

in-house

method

upon

serial

dilution

of

RNA

extracts

Despite

having

comparable

sensitivity

amplifications

were

rapidly

lost

when

a

larger

fragment

was

amplified

using

the

ViroSeq

assay

The

main

objective

of

our

study

was

to

evaluate

the

amplification

success

rate

from

DBSs

stored

at

°C

for

a

prolonged

period

of

time

We

used

an

in-house

assay

validated

for

subtype

B

since

our

study

population

included

patients

infected

with

subtype

B

viruses

It

is

important

to

note

however

that

information

on

the

performance

of

our

assay

with

non-B

subtypes

is

limited

to

a

small

number

of

specimens

Therefore

we

caution

that

resistance

testing

from

DBSs

collected

in

areas

with

prevalent

non-B

subtypes

will

require

in-house

assays

appropriately

validated

for

other

HIV

subtypes

The

ability

to

collect

blood

samples

on

filter

paper

represents

an

advantage

for

HIV

drug

resistance

surveillance

and

monitoring

particularly

in

areas

that

lack

the

appropriate

infrastructure

for

plasma

processing

and

transport

Much

effort

is

currently

underway

to

define

the

best

conditions

that

will

facilitate

resistance

testing

from

DBSs

including

the

most

appropriate

storage

temperature

and

time

The

findings

reported

here

suggest

that

°C

may

represent

a

feasible

storage

temperature

for

long-term

storage

of

DBSs

and

add

to

the

promise

of

DBSs

as

a

convenient

specimen

type

for

drug

resistance

testing

Funding

Work

at

the

CDC

was

done

with

intramural

funding

Work

at

Hospital

Carlos

III

was

supported

by

grants

from

the

Fondo

de

Investigación

Sanitaria

-LRB-

FIS

and

-RRB-

Transparency

declarations

to

declare

Disclaimers

The

findings

and

conclusions

in

this

report

are

those

of

the

authors

and

do

not

necessarily

represent

the

views

of

the

Centers

for

Disease

Control

and

Prevention

Use

of

trade

names

is

for

identification

only

and

does

not

constitute

endorsement

by

the

U.S.

Department

of

Health

and

Human

Services

the

Public

Health

Service

or

the

Centers

for

Disease

Control

and

Prevention

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

resistance testing
viroseq assay
903 filter paper


Crystal Structure of an Active Form of Human MMP-1 The extracellular matrix is a dynamic environment that constantly undergoes remodelling and degradation during vital physiological processes such as angiogenesis , wound healing , and development . Unbalanced extracellular matrix breakdown is associated with many diseases such as arthritis , cancer and fibrosis . Interstitial collagen is degraded by matrix metalloproteinases with collagenolytic activity by MMP-1 , MMP-8 and MMP-13 , collectively known as the collagenases . Matrix metalloproteinase 1 -LRB- MMP-1 -RRB- plays a pivotal role in degradation of interstitial collagen types I , II , and III . Here , we report the crystal structure of the active form of human MMP-1 at 2.67 Å resolution . This is the first MMP-1 structure that is free of inhibitor and a water molecule essential for peptide hydrolysis is observed coordinated with the active site zinc . Comparing this structure with the human proMMP-1 shows significant structural differences , mainly in the relative orientation of the hemopexin domain , between the pro form and active form of the human enzyme . Introduction Connective tissue remodeling is a complex process involving a plethora of cytokines , growth factors , and turnover of extracellular matrix -LRB- ECM -RRB- . The main enzymes that degrade ECM molecules are matrix metalloproteinases -LRB- MMPs -RRB- , which are also known as matrixins . Under normal physiological conditions , MMP activity can be regulated at various stages : during transcription , proteolytic processing of their inactive pro forms , zymogens , as well as by inhibition of enzyme activity by endogenous inhibitors such as tissue inhibitors of metalloproteinases or TIMPs .1,2 These enzymes have a similar domain structure : an N-terminal signal sequence to target for secretion , a pro-peptide domain to maintain latency , a catalytic domain containing the catalytic zinc , a linker region , and a C-terminal four-bladed propeller structure called the hemopexin domain . Some of the MMPs have additional domains , e.g. the fibronectin repeats in gelatinases . These domains are important in substrate recognition and in inhibitor binding .3 The human MMP family to date comprises of about 23 enzymes that are classified based on their preferred substrate and cellular localisation : collagenases , gelatinases , stromelysins , elastase , membrane-type MMPs and so forth .4 Collagenases -LRB- MMP-1 , MMP-8 and MMP-13 -RRB- are the key enzymes that are capable of cleaving interstitial fibrillar collagen . Apart from these enzymes MMP-2 -LRB- gelatinase A -RRB- and MMP-14 -LRB- MT1-MMP -RRB- are also able to initiate the breakdown of collagen fibrils .5,6 Interstitial collagens I , II and III are triple-helical proteins that are the essential structural components of all connective tissues such as the cartilage , bone , skin , tendons and ligaments . These extracellular glycoproteins provide scaffolding of the tissue and play an important role in cellular processes such as cell migration , proliferation and differentiation . Physiological collagenolysis is integral to several biological processes such as embryogenesis , tissue repair and remodeling , angiogenesis , organ morphogenesis and wound healing .7,9 The collagenases cleave the triple-helical collagen approximately three-quarters away from the N terminus of the substrate , resulting in three-quarters and one quarter length fragments that are unstable at body temperature and undergo denaturation , rendering them susceptible to other non-specific tissue proteinases . However , under aberrant circumstances degradation of collagen results in pathological conditions such as cancer , atherosclerosis , arthritis , aneurysm and fibrosis .8,10 The proteolytic activity of the enzyme resides in the catalytic domain but it requires the hemopexin domain in order to cleave the three chains of the triple-helical collagen .11 The crystal structure of porcine MMP-1,12 determined sometime ago , revealed details of the active site structure and the specificity pocket but this structure does not shed any light on how collagenolytic MMPs can cleave the triple-helical collagen . Recently , the X-ray structure of human proMMP-1 -LRB- MMP-1 zymogen -RRB- was elucidated .13 The structure revealed the interaction between the pro-peptide and the hemopexin domain of the enzyme , which results in a `` closed '' conformation of the zymogen in contrast to the `` open '' conformation of catalytic domain of the active MMP-1 . The three-dimensional structure of the human MMP-1 -LRB- E200A -RRB- , an active site mutant , reported here takes us a step closer towards a more complete understanding of the interaction of the collagenases with the triple-helical collagens . It reveals new features of the active protease and provides a platform for understanding the structural changes that accompany zymogen activation . Results Overall structure The structure of human MMP-1 -LRB- E200A -RRB- was determined at 2.67 Å resolution with two monomers -LRB- chains A and B -RRB- in the asymmetric unit of the trigonal space group , P3221 -LRB- see Table 1 for crystallographic statistics -RRB- . The overall domain structure of human MMP-1 is similar to that of the previously solved full-length enzymes -LRB- Figure 1 -RRB- . The structure comprises of the N-terminal catalytic domain , the linker region and the C-terminal hemopexin domain . The catalytic domain of one monomer contacts the hemopexin domain of the other monomer . Interestingly , the contact site used by the two monomers in the asymmetric unit to form the dimer is not the same as the dimerisation site observed in the proMMP-1 structure13 or that for the MMP-9 hemopexin domain .14 This indicates that the dimerisation mechanism is perhaps not a general rule of thumb for the hemopexin domains and is most probably a crystallisation artefact . Monomers A and B deviate from each other with an overall r.m.s. deviation of 0.87 Å -LRB- for 367 Cα atoms -RRB- . The dimer is stabilised by eight hydrogen bonds and 54 van der Waals contacts -LRB- Table 2 -RRB- . These contacts are facilitated by a total of 33 residues -LRB- 18 residues from monomer A and 15 residues from monomer B -RRB- . Only nine amino acid residues are common to both the monomers : Pro104 , Asp105 , Leu106 , Arg183 , Trp184 , Thr185 , Val300 , Phe301 and Gln335 . Apart from the asymmetry seen in the residues that participate from each monomer at the dimer interface , we also observe a non-symmetric interaction pattern . However , it is believed that this dimer is not physiologically relevant , as we found that human MMP-1 is a monomer in solution -LRB- R.V. & H. N. , unpublished results -RRB- . Catalytic domain The structure of MMP-1 catalytic domain is very similar to those of other MMPs . The metalloproteinase domain is about 160 amino acid residues in length with the catalytic zinc ion residing in the C-terminal segment of this domain . The catalytic fragment of the protease consists of three α-helices and a highly twisted five-stranded β-sheet . The active site zinc is bound in the sequence HELGHXXGXXH by the three His residues : His199 , His203 , His209 and a water molecule at the active site cleft -LRB- Figure 2 -RRB- . This is the first MMP-1 structure where a water molecule essential for peptide hydrolysis is observed at the active site because all the previous structures of the metalloproteinase domain of MMP-1 are in the inhibited state of the enzyme . Also observed in this domain is the salt bridge between the ammonium group of the N-terminal , Phe81 and the carboxylate group of the side-chain of Asp232 . The generation of the correct N terminus -LRB- Phe81 -RRB- in the activation process of proMMP-1 is crucial for the enzyme to have full activity against collagen .15 If the N terminus is either longer or shorter the activity against collagen drops to 30 -- 40 % .15 Only with the correct N terminus the formation of the salt bridge is possible , and this stabilises the structure of the N terminus as was originally shown for MMP-8.16,17 Alignment of the pro-enzyme -LRB- 1SU3 -RRB- structure with that of the mature enzyme -LRB- 2CLT here -RRB- shows Phe81 to have moved some 17 Å from its original position in the zymogen form of collagenase-1 -LRB- Figure 3 -LRB- a -RRB- -RRB- . The structure of proMMP-3 -LRB- PDB code : 1SLM -RRB- 18 was aligned as well to help calculate the displacement of Phe81 . The first seven N-terminal residues were not observed in the proMMP-1 structure because of lack of proper visible electron density .13 This deviation gradually decreases and by residue Pro88 the two structures are in register and remain remarkably similar throughout the catalytic domain . There are only slight differences , if any , in the side-chain orientation of the N-terminal residues in the catalytic domain cleft -LRB- Figure 3 -LRB- b -RRB- -RRB- . The structural zinc exhibits tetrahedral coordination facilitated by residues His149 , Asp151 , His164 and His177 . The catalytic domain of MMP-1 also contains three calcium-binding sites . Either four or five liganding residues coordinate all the calcium ions , except one in the catalytic domain of monomer A -LRB- Table 3 -RRB- . One calcium ion packs the S-loop -LRB- between strands s3 and s4 -RRB- against the liganding residues from strand s5 . The second calcium ion of the catalytic domain is sandwiched between strand s3 and the loop containing strands s4 and s5 . The third calcium site is determined by the presence of the critical residue , Asp105 that comes from the loop following strand s1 . The other residues -LRB- Glu180 and Glu182 -RRB- that provide coordination to this calcium ion come from the loop following strand s5 . One interesting feature observed in this crystal structure by virtue of dimerisation is that residue Gln335 from monomer B contributes the fourth coordinating ligand for this calcium ion . This feature , however , is not replicated in the corresponding calcium-binding site in monomer B. Details of the metal site geometries can be found in Table 3 . Linker region In MMPs the catalytic domain is followed by a stretch of 15 -- 65 amino acid residues referred to as the linker or the hinge region . This region is rich in proline residues and replacement of those with alanine drastically reduced the collagenolytic activity of MMP-8 -LRB- neutrophil collagenase -RRB- ,19 indicating that the presence of the correct linker structure is important for collagenolysis . This region in MMP-1 with 16 residues is well defined in the present structure unlike that in the full-length crystal structure of porcine collagenase . The conformation of the linker region is quite similar in both proMMP-113 and the present MMP-1 structures -LRB- Figure 4 -LRB- a -RRB- -RRB- . The residues of the linker region make extensive contacts -LRB- six hydrogen bonds and 73 van der Waals contacts ; Table 4 -RRB- with both the catalytic and the hemopexin domain of the enzyme . These interactions stabilise the domain arrangement in MMP-1 , which is required for the concerted action of the two domains . Comparing the interactions of the linker region with the catalytic and the hemopexin domain in both the pro-enzyme and the active enzyme did not reveal many differences . Most of the interactions seen in proMMP-1 -LRB- seven hydrogen bonds and 71 van der Waals contacts -RRB- are still observed in the present structure , indicating that the activation of the enzyme does not result in structural changes and the overall structure of the areas around the linker region are mostly conserved . Interestingly though , the hydrogen-bonding interactions -LRB- Table 4 -RRB- within this region are quite different between the two forms of the molecule . There are a total of seven hydrogen bonds within the linker region of active MMP-1 -LRB- Figure 4 -LRB- b -RRB- -RRB- compared to the five observed in proMMP-1 -LRB- Figure 4 -LRB- c -RRB- -RRB- , with only two bonds common to both structures This difference in the number of hydrogen bonds is perhaps not of much significance when seen in the light of the extensive contacts this region makes with the other two domains . Hemopexin-like domain The hemopexin domain starts with Cys259 and forms a complete circle by joining to Cys447 in a disulphide bond that connects blade bI with blade bIV , giving this domain the characteristic four-bladed β-propeller structure . Each blade starts near the periphery with either the motif DAA or DAX , in which the Asp residues -LRB- Asp266 , Asp359 and Asp408 -RRB- coordinate the central calcium ion through their carbonyl oxygen atom . Glu310 provides the fourth coordination thus completing the acidic patch at the entrance of the central , solvent-accessible channel -LRB- Table 3 -RRB- . The side-chains of these residues form salt bridges to the neighbouring β-strands holding the entrance of the central channel together . Three water molecules are found trapped in the centre of this channel . These , however , are not involved in the coordination geometry of the calcium ion at the tunnel centre . Two of the water molecules are at positions corresponding to the sodium and chloride ion in the proMMP-1 structure .12 The water molecule corresponding to the sodium ion is at hydrogen-bonding distances to the carbonyl oxygen atom of Ile268 , Ala312 , Ala361 and Val410 . The same can not be said for the one corresponding to the chloride ion , which does not make any hydrogen bonds with the main-chain amides of the residues mentioned above . It is very likely that the presence of these ions is a consequence of the crystallisation conditions rather than a stability requirement for the hemopexin-like domain . Comparison with the porcine MMP-1 and the human proMMP-1 structures Full-length human MMP-1 , human proMMP-113 and porcine MMP-112 were superimposed on the basis of the catalytic domain of the three structures . Alignments were also done for just the hemopexin domain of the molecules . As expected both types of alignments gave the same outcome whereby the active form of the enzyme -LRB- human and porcine -RRB- were closer in their structure as opposed to the proMMP-1 structure . The average r.m.s. deviation over 367 Cα atoms of the porcine and human MMP-1 is ∼ 1.4 Å , whereas the r.m.s. deviation when aligning the full-length active MMP-1 with the proMMP-1 structure is 1.6 Å . If , however , we take only the hemopexin-like domain into consideration , then the conformational differences that lie within this domain are brought to light -LRB- Figure 5 -LRB- a -RRB- -RRB- . The superimposed porcine and human MMP-1 hemopexin-like domains vary by 1.2 Å -LRB- maximum displacement of 5.2 Å -RRB- . This value increases to 1.7 Å when superimposing the hemopexin-like domains of the active human MMP-1 and the human proMMP-1 . A considerable change in conformation is reflected in the residues that interact with the pro-domain . Most of these differences are by virtue of the Phe289-Tyr290-Pro291 region of the hemopexin-like domain . Table 5 shows these residues , their interactions and the shift in Cα position observed when the pro and active forms of human MMP-1 were superimposed . The largest movement of 16 Å is observed for the residue , Phe289 -LRB- Figure 5 -LRB- b -RRB- -RRB- . Such a perturbation in structural conformation is not evident in the catalytic domain of the enzyme -LRB- Table 5 -RRB- . It seems like Arg281 acts as a pivot around which the hemopexin-like domain undergoes displacement upon activation as suggested by Jozic et al. 13 One region in the catalytic domain that generates interest is the region between Tyr218 and Tyr221 . The shift in the Cα position between the active and the proMMP-1 is not as dramatic as that observed for the Phe289-Tyr290-Pro291 region but they are involved in several interactions with the pro domain around the cysteine-switch region . As a result when the pro-enzyme undergoes activation , this region of the catalytic domain opens wider and further exposes the crucial cis-configured Glu190-Tyr191 peptide in MMP-1 . Discussion Reconciliation of structural findings : pro-collagenase versus active collagenase MMPs are multi-domain enzymes that consist of a pro domain , catalytic domain , a linker region and a hemopexin-like domain . Cleavage of the pro domain leads to a substantial rearrangement of the N-terminal residues 81 -- 88 -LRB- Figure 3 -LRB- a -RRB- -RRB- . The activation process swings Phe81 towards the proteinase domain and terminates with a salt link between the amine of Phe81 and the carboxylate side-chain of Asp232 . This interaction imparts several-fold greater enzymatic activity to MMP-1 than those with either an extended or a shorter N terminus , which lack the salt linkage .17 Previously an inhibitor-free structure of the catalytic domain of MMP-1 was reported .20 This structure was , however , unique in the sense that the N-terminal Leu-Thr-Glu-Gly -LRB- 83 -- 86 -RRB- residues of one molecule occupied the active site of the other molecule thus forming an unnatural inhibited complex , which should not really be considered as an inhibitor-free structure . The present structure on the other hand stands unique in being elucidated in the absence of an inhibitor . This is the first unliganded structure of the full-length MMP-1 where a water molecule is seen at the active site providing the fourth ligand for the catalytic zinc in the tetrahedral coordination sphere -LRB- Figure 2 -RRB- . It must be mentioned here that the present structure is an active site mutant where the catalytic glutamate residue has been mutated to an alanine . It is likely that the scissile peptide bond and the catalytic glutamate -LRB- Glu200 -RRB- flank the active site water molecule on either side . The favourable hydrogen bond between Glu200 and the water molecule would make it more nucleophilic . The strongly polarised carbonyl group of the scissile bond -LRB- by virtue of its interaction with the catalytic zinc -RRB- would face a properly oriented water molecule . An unstable transition state results in the transfer of one water proton to the leaving nitrogen -LRB- the amino group of the scissile bond -RRB- via the Glu200 carboxylate shuttle . This is followed by the cleavage of the peptide bond and concomitant shuttle of another water proton to the amino group . This hypothesis will , however , need to be substantiated with a transition state structure . The linker region is considered to play an important role in collagenolysis ,19,21 but its exact role is not clear . The structures of both proMMP-1 and active MMP-1 have revealed the residues of this region to be in close contact with the catalytic and the hemopexin-like domain . Comparative analysis of the extensively similar contacts made by the residues of the hinge region reveal that despite being highly exposed with no secondary structure , the conformation of the linker peptide is perhaps a signature of its sequence and does not depend on the crystal packing -LRB- Figure 4 -LRB- a -RRB- -RRB- . Mutagenesis studies of this region result in decreased collagenolytic activity of MMP-121 and MMP-8.19 This may be due to structural changes in this region affecting the interactions with the catalytic and hemopexin domains that are required for collagenolysis .22 The cleavage of the pro domain to form the active enzyme is accompanied by major conformational rearrangement in the residues that interact with the pro domain . Residues from both the catalytic domain and the hemopexin-like domain make interactions with the pro domain residues . The effects on the overall backbone structure of the catalytic domain is minimal ; the effects on the hemopexin-like domain is , however , quite dramatic -LRB- Figure 5 -LRB- a -RRB- -RRB- . The Phe289-Tyr290-Pro291 region of the hemopexin-like domain undergoes the most significant conformational change -LRB- Figure 5 -LRB- b -RRB- -RRB- . In essence , the hemopexin-like domain undergoes a major displacement towards the catalytic domain , thus widening the cleft between the proteinase domain and the hemopexin-like domain on the active site face of the enzyme . This altered configuration makes the active site residues and the RWTNNFREY -LRB- residues 183 -- 191 -RRB- interface , a segment critical for collagenolysis ,23 more accessible for native collagen . Implications for collagenolysis Despite growing awareness of the importance of collagen-recognising determinants it is still unclear as to which part of the collagenase molecule makes the first contact with the triple-helical collagen . Several hypotheses have been suggested to try and explain the steps involved in collagenolysis -LRB- the most recent by Jozic et al. 13 -RRB- . Docking studies were performed using a single-stranded , collagen α1 -LRB- I -RRB- - like 15mer peptide with the Gly-Ile cleavage site incorporated in an attempt to better understand the functioning of the active enzyme . The peptide was modelled on the known structure of one of the chains of the triple-helical collagen -LRB- PDB code : 1BKV -RRB- .24 Several solutions were generated . We used localised interactions as our guide to help us pick one possible solution : -LRB- a -RRB- contact of the carbonyl group of the Gly-Ile bond of the peptide with the active site determinants , especially the catalytic zinc ; and -LRB- b -RRB- contact with the critical cis-configured Glu190-Tyr191 peptide . This solution revealed the peptide to be aligned to the continuous bulge-edge strand -LRB- Gly160-Phe166 -RRB- and the wall-forming segment -LRB- Pro219-Phe223 -RRB- in an extended manner . This solution , however , upon energy minimisation moved outside of the coordination sphere of the catalytic zinc but it was still close enough to be polarised by the catalytic zinc . Since the peptide was only 15 amino acid residues long , we could not see any interactions with the C-terminal hemopexin-like domain . It has been proposed that the triple-helical collagen would run via the Glu190-Tyr191 cis-peptide exosite making extensive interactions with the residues of the hemopexin-like domain .22 The structure of the active form of human MMP-1 has provided us insights on the conformational changes that occur upon activation of the pro-enzyme and these are valuable clues towards understanding the mechanism of collagenolysis . A better understanding of the mechanism of collagen cleavage will assist towards design of inhibitors that would specifically interfere with collagenolysis without affecting -LRB- beneficial -RRB- the cleavage of other substrates . Experimental Procedures Cloning , protein expression and protein purification The catalytically inactive mutant proMMP-1 -LRB- E200A -RRB- was cloned , overproduced , refolded , and purified as described .22 A catalytic site mutant of this enzyme was chosen in order to prevent autocatalytic cleavage during crystallisation . The mutant zymogen was activated as described22 at a final concentration of 554 μM in the presence of 1:80 molar ratio of MMP-3 lacking the hemopexin-like domain -LRB- MMP-3ΔC -RRB- and 1 mM 4-aminophenylmercuric acetate for 90 min at 37 °C . The activation mixture was directly applied to a Sephadex S-200 gel filtration column -LRB- diameter 26 mm , length 900 mm -RRB- in 50 mM Tris -- HCl -LRB- pH 7.5 -RRB- , 150 mM NaCl , 10 mM CaCl2 , 0.02 % -LRB- w/v -RRB- sodium azide , to separate the `` active '' form of MMP-1 -LRB- E200A -RRB- from MMP-3ΔC , 4-aminophenylmercuric acetate , and remnants of the pro-peptide . Fractions containing MMP-1 -LRB- E200A -RRB- were pooled and concentrated using a Vivacell 250 ml with a 5 kDa cutoff membrane , followed by a Vivaspin 20 with a 5 kDa membrane -LRB- Vivascience -RRB- . The protein was stored at room temperature as incubation at 4 °C resulted in precipitation . Crystallisation , data collection and processing MMP-1 -LRB- E200A -RRB- was crystallised using the hanging drop vapour-diffusion method . The protein -LRB- 2 μl at a concentration of 21 mg/ml -RRB- was mixed with 2 μl of the reservoir solution containing 0.1 M Tris -LRB- pH 7.5 -RRB- , 1.5 M ammonium formate and 10 % -LRB- w/v -RRB- polyethylene glycol -LRB- PEG -RRB- 8000 . Crystals appeared and grew to their maximum size within two weeks at 16 °C . A cryoprotectant solution prepared by supplementing the reservoir with 25 % -LRB- v/v -RRB- glycerol enabled the crystals to be flash-frozen in liquid nitrogen . Flash-cooled MMP-1 -LRB- E200A -RRB- crystals were used to collect diffraction data to a resolution of 2.67 Å on PX 14.1 at the Synchrotron Radiation Source , Daresbury -LRB- UK -RRB- . The data were processed and scaled using HKL2000 .25 The crystals belong to the space group P3221 , with cell dimensions of a = b = 138.48 Å and c = 110.05 Å . There are two monomers in the asymmetric unit with solvent content of about 60 % . Data reduction using the program TRUNCATE26 estimated an overall B-factor of 63.1 Å2/Da from the Wilson plot . Details of the data processing statistics are presented in Table 1 . Structure determination The solution for the MMP-1 -LRB- E200A -RRB- structure was found using the program PHASER .26 Human proMMP-1 -LRB- PDB code : 1SU3 -RRB- 13 was used as the search model with the pro-peptide domain removed from the structure . Log-likelihood gain -LRB- LLG -RRB- of 161 for the first solution increased to an LLG value of 1091 when the second monomer in the asymmetric unit was located indicating the solutions were indeed the right ones . Refinement Crystallographic refinement was carried out using the program CNS27 at 2.67 Å resolution against 85.1 % of the measured data . A test set of random reflections of 2.2 % was excluded from the full data set for cross-validation purposes by calculating the free R-factor -LRB- Rfree -RRB- to monitor refinement trend .28 Initial round of refinement with the two monomers found by PHASER26 resulted in an Rcryst of 45.1 % and an Rfree of 50.6 % . Once the six N-terminal residues in both the monomers were built in and all the ions -LRB- two zinc ions and four calcium ions per monomer -RRB- were added and the model subjected to simulated annealing , the Rcryst and the Rfree dropped to 31 % and 38 % , respectively . Iterative cycles of refinement -LRB- energy minimisation , simulated annealing and individual temperature factor -LRB- B-factor -RRB- refinement -RRB- using CNS27 and model building with reference to 2Fo-Fc and Fo-Fc maps using the program Coot29 progressively improved the phases . In the final stages of refinement , water molecules with peaks greater than 3σ in the Fo-Fc maps and those within hydrogen bonding distances from appropriate atoms were incorporated into the structure . The final refined structure at 2.67 Å resolution has an Rcryst of 22.3 % and an Rfree of ∼ 26 % . Both monomers , chains A and B have the full complement of the amino acids : from Phe81 to Cys447 . Residues 117 , 356 , 369 , 385 , 386 and 424 of chain A and residues 257 , 272 , 287 , 424 and 446 of chain B have been modelled as alanine residues because of the lack of visible electron density beyond Cβ atom . Analysis of the Ramachandran plot using the program PROCHECK26 indicated that ∼ 81 % of the residues are in the most favourable region of the ϕ-φ plot and about 17 % lie in the additional allowed region . The structure also consists of 198 water molecules . Details of the refinement statistics are given in Table 1 . Protein Data Bank accession codes The atomic coordinates and the structure factors of the human MMP-1 -LRB- E200A -RRB- have been deposited with the RCSB Protein Data Bank -LRB- accession codes 2CLT and R2CLTSF , respectively -RRB- .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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 O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mmp, matrix metalloproteinase
ecm, extracellular matrix
fibroblast collagenase
x-ray crystallography
inhibitor-free