Collagens
are
the
major
structural
proteins
in
animal
tissues.
Their
degradation
is
essential
in
embryogenesis
and
development,
while
unbalanced
collagen
breakdown
is
seen
in
diseases
such
as
arthritis,
atherosclerosis
and
cancer.
Fibril-­forming
collagens
I,
II
and
III
consist
of
three
polypeptide
chains
forming
a
triple
helix,
which
is
resistant
to
cleavage
by
most
proteases.
Collagenases
of
the
matrix
metalloproteinase
family
(MMP-­1,
MMP-­8,
and
MMP-­13)
degrade
fibrillar
collagens
by
locally
unwinding
the
helix,
followed
by
cleavage
into
¼
and
¾
fragments.
They
comprise
two
domains,
the
catalytic
(Cat)
domain
and
the
hemopexin
(Hpx)
domain,
which
are
connected
via
a
flexible
linker.
Both
domains
are
essential
for
collagenolysis,
but
the
exact
sites
of
collagenase-­collagen
interactions
and
how
they
unwind
collagen
remain
elusive.
This
thesis
addresses
the
roles
of
individual
collagenase
domains,
and
the
sites
in
both
the
enzyme
and
the
substrate
that
are
involved
in
collagen
binding
and
unwinding,
focusing
on
the
fibril-­forming
collagens
and
human
MMP-­1
as
a
prototype.
MMP-­1
bound
to
immobilised
collagen
I
with
markedly
higher
affinity
than
its
Hpx
domain
alone.
The
Cat
domain
alone
failed
to
bind
to
collagen,
but
in
the
full-­length
enzyme
it
participated
in
collagen
binding.
Above
25°C
the
two-­
domain
binding
involved
the
catalytic
site
cleft.
Triple-­helical
peptide
(THP)
Toolkits
of
collagens
II
and
III
were
screened
for
MMP-­1
binding,
and
the
collagenase
binding
motif
has
been
established.
It
contains
two
hydrophobic
residues
within
a
9
residue
distance.
Finally,
hydrogen/deuterium
exchange
mass
spectrometry
(H/DXMS)
experiments
indicated
two
potential
collagen
binding
sites:
285-­316
and
349-365
in
the
Hpx
domain,
and
suggested
a
possibility
of
a
dynamic
interaction
of
the
collagenase
N-­terminus
with
collagen.
These
results
imply
that
the
two
domains
of
collagenase
bind
to
collagen
in
a
cooperative
manner.
Based
on
the
THP
binding
and
H/DXMS
data
a
3D
model
of
collagenase-­collagen
interaction
has
been
proposed.
It
assumes
that
collagenase
utilises
hydrophobic
interactions
to
unwind
the
collagen
helix
via
perturbation
of
the
hydrogen-­bond
network
which
stabilises
the
helix.