Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1.
ABSTRACT Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.
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ABSTRACT: The molecular conformation of the collagen triple helix confers strict amino acid sequence constraints, requiring a (Gly-X-Y)(n) repeating pattern and a high content of imino acids. The increasing family of collagens and proteins with collagenous domains shows the collagen triple helix to be a basic motif adaptable to a range of proteins and functions. Its rodlike domain has the potential for various modes of self-association and the capacity to bind receptors, other proteins, GAGs, and nucleic acids. High-resolution crystal structures obtained for collagen model peptides confirm the supercoiled triple helix conformation, and provide new information on hydrogen bonding patterns, hydration, sidechain interactions, and ligand binding. For several peptides, the helix twist was found to be sequence dependent, and such variation in helix twist may serve as recognition features or to orient the triple helix for binding. Mutations in the collagen triple-helix domain lead to a variety of human disorders. The most common mutations are single-base substitutions that lead to the replacement of one Gly residue, breaking the Gly-X-Y repeating pattern. A single Gly substitution destabilizes the triple helix through a local disruption in hydrogen bonding and produces a discontinuity in the register of the helix. Molecular information about the collagen triple helix and the effect of mutations will lead to a better understanding of function and pathology.Advances in protein chemistry 02/2005; 70:301-39. · 3.20 Impact Factor
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ABSTRACT: The degradation of interstitial collagens I, II and III is an integral part of many biological events such as embryonic development, organ morphogenesis, tissue remodelling and repair, and in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis, but these collagens are resistant to most proteolytic enzymes due to their triple helical structures. In vertebrates, the enzymes that degrade interstitial collagens are collagenases that are members of the matrix metalloproteinase (MMP) family, and cathepsin K, a lysosomal cysteine proteinase. Non-vertebrate collagenases include collagenolytic serine proteinases in crustacea and Clostridium histolyticum collagenases, also called “bacterial collagenases.” This chapter describes the unique properties of these collagenolytic proteinases and the current proposal as to how they recognize triple helical structures and cleave them. Potential enzymatic pathways that may be involved in the degradation of collagen fibrils are also discussed.07/2011: pages 95-122;
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ABSTRACT: It is 40 years since the first member of what came to be known as the matrix metalloproteinase (MMP) family was described. Structural, molecular and biochemical approaches have subsequently contributed to piecing together the puzzle of how MMPs work, and how they contribute to various disease processes.Nature Reviews Molecular Cell Biology 04/2002; 3(3):207-14. · 37.16 Impact Factor
Structural insights into triple-helical collagen cleavage
by matrix metalloproteinase 1
Szymon W. Mankaa, Federico Carafolib, Robert Vissea, Dominique Bihanc, Nicolas Raynalc, Richard W. Farndalec,
Gillian Murphyd, Jan J. Enghilde, Erhard Hohenesterb,1, and Hideaki Nagasea,1
aNuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford, London W6
8LH, United Kingdom;bDepartment of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom;cDepartment of Biochemistry, University of
Cambridge, Cambridge CB2 1QW, United Kingdom;dDepartment of Oncology, Cancer Research UK Cambridge Research Institute, University of Cambridge,
Cambridge CB2 0RE, United Kingdom; andeDepartment of Molecular Biology and Genetics, University of Aarhus, DK-8000 Aarhus, Denmark
Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved June 11, 2012 (received for review March
Collagenases of the matrix metalloproteinase (MMP) family play
major roles in morphogenesis, tissue repair, and human diseases,
but how they recognize and cleave the collagen triple helix is not
fully understood. Here, we report temperature-dependent binding
the cooperative action of its catalytic and hemopexin domains.
Contact between the two molecules was mapped by screening the
Collagen Toolkit peptide library and by hydrogen/deuterium ex-
change. The crystal structure of MMP-1(E200A) bound to a triple-
helical collagen peptide revealed extensive interactions of the 115-
Å–long triple helix with both MMP-1 domains. An exosite in the
hemopexin domain, which binds the leucine 10 residues C-terminal
to the scissile bond, is critical for collagenolysis and represents a
unique target for inhibitor development. The scissile bond is not
1 structure or the exosite interactions, by axial rotation of the colla-
gen homotrimer. Interdomain flexing of the enzyme and a localized
excursion of the collagen chain closest to the active site, facilitated
state of collagenolysis.
extracellular matrix|X-ray crystallography|protease
tendon, and blood vessels (1). They consist of three α chains with
repeating Gly-X-Y triplets (X and Y are often proline and
hydroxyproline, respectively) that intertwine each other to form
a triple helix of ∼300 nm in length (2). Interstitial collagens are
resistant to most proteolytic enzymes, but vertebrate collage-
nases cleave them at a single site approximately three-quarters of
the way from the N terminus of the triple helix, thus initiating
collagenolysis (3). Owing to this unique activity, collagenases
play important roles in embryo development, morphogenesis,
tissue remodeling, wound healing, and human diseases, such as
arthritis, cancer, and atherosclerosis (4, 5).
Matrix metalloproteinase 1 (MMP-1) is a typical vertebrate
collagenase (3). It consists of an N-terminal catalytic (Cat) do-
main containing an active-site zinc ion and a C-terminal hemo-
pexin (Hpx) domain comprised of a four-bladed β-propeller,
which are connected by a linker region (6, 7). Although the Cat
heat-denatured collagen (gelatin), its activity on native triple-
helicalcollagen isnegligible. Thecombination ofthe CatandHpx
domains is required for MMP-1 to be able to degrade native
collagen, and the same is true for all other collagenolytic MMPs,
namely MMP-2, MMP-8, MMP-13, and MMP-14 (3). How col-
lagenases interact with collagen and how the Hpx domain endows
these enzymes with collagenolytic activity is not clearly un-
derstood. Another enigma of collagenolysis became apparent
when the crystal structures of MMP-1Cat (8–10), MMP-8Cat (11,
12),andfull-length pig MMP-1(6)weresolved: Thecatalytic cleft
he interstitial collagens I, II, and III are the major structural
proteins in connective tissues such as skin, bone, cartilage,
of these enzymes is too narrow to accommodate collagen in its
native triple-helical conformation. A number of hypotheses have
been proposed to explain how collagenases may destabilize and
cleavetriple-helical collagen (13–16),andwehave experimentally
demonstrated that MMP-1 unwinds triple-helical collagen locally
before peptide bond hydrolysis (17, 18). A recent study mapped
the interaction sites in MMP-1 and in triple-helical collagen by
using NMR and proposed a model of collagen binding and un-
winding (19). However, because the interactions between MMP-1
andcollagen were not observed directly, a number ofassumptions
had to be made to derive a mechanism of collagenolysis.
In this study, we have combined biochemical experiments with
crystallographic structure determination to show how MMP-1
recognizes its unique cleavage site in interstitial collagens. Our
results suggest that collagenolysis relies on multiple exosite
interactions, which not only serve to position the scissile bond
near the active site, but also assist in the local unfolding of the
collagen triple helix that is required for cleavage to occur.
Cat and Hpx Domains of MMP-1 Cooperate in Collagen Binding. We
first examined a catalytically inactive MMP-1(E200A) mutant
(residue numbering based on the proMMP-1 sequence) and its
individual domains for binding to native collagen I. Full-length
MMP-1(E200A) showed saturable collagen binding with an ap-
parent KDof 0.4 μM, whereas the catalytic domain Cat(E200A)
showed no detectable binding and the Hpx domain bound only
weakly (Fig. 1A). The Cat(E200A) domain did not bind to
collagen I even when it was added together with increasing con-
centrations of the Hpx domain (SI Appendix, Fig. S1A), suggest-
ing that the Cat and Hpx domains bind collagen cooperatively
only when they are linked together.
The binding of MMP-1(E200A) to collagen increased with
temperature until collagen denatured above 37 °C (Fig. 1B),
indicating that MMP-1 prefers a looser triple helix but not
denatured collagen. The temperature-dependent enhancement
of MMP-1(E200A) binding to collagen was substantially reduced
by the active-site inhibitor GM6001 (Fig. 1C). This effect was
Author contributions: S.W.M., R.W.F., G.M., J.J.E., E.H., and H.N. designed research; S.W.M.,
F.C., R.V., D.B., and E.H. performed research; D.B., N.R., R.W.F., G.M., and J.J.E. contributed
new reagents/analytic tools; S.W.M., F.C., R.V., R.W.F., G.M., J.J.E., E.H., and H.N. analyzed
data; and S.W.M., E.H., and H.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 4AUO).
1To whom correspondence may be addressed. E-mail: email@example.com
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 31, 2012
| vol. 109
| no. 31
not observed below 10 °C, where little collagen cleavage occurs
(17). No significant temperature-dependent increase in collagen
binding was observed with either the Cat(E200A) or Hpx domain
alone (SI Appendix, Fig. S1 B and C). These results suggest that
the active site cleft of MMP-1 participates in collagen binding
at body temperature by stabilizing and/or inducing a looser
MMP-1–Binding Motif in Interstitial Collagens. To identify the col-
lagen residues interacting with MMP-1, we screened the Colla-
gen Toolkit library of triple-helical peptides encompassing the
entire collagen II sequence (20) for binding to MMP-1(E200A).
Only one peptide, Col II-44, starting at the collagenase cleavage
site G∼LAGQR... (∼ indicates the scissile bond) was recog-
nized by the enzyme (Fig. 2). Col II-43, which also contains the
cleavage site but with fewer C-terminal residues, did not bind
MMP-1(E200A). Further experiments with truncated and ala-
nine-substituted peptides identified the leucines at the P1′ and
P10′ positions (nomenclature after Schechter and Berger; ref.
21) of collagen as important for MMP-1(E200A) binding (SI
Appendix, Fig. S2A). A sequence alignment showed that the
leucines at these two positions are conserved in all α chains of
collagens I, II, and III from different species (SI Appendix, Fig.
S2B). The consensus sequence for collagenase binding is G∼(L/
I)(A/L)-GXY-GXY-GL(O/A) (P1′ and P10′ positions are bold;
O, hydroxyproline). This motif is unique and not found else-
where in collagens I, II, and III.
Mapping of the Collagen-Binding Regions of MMP-1 in Solution. We
then used hydrogen/deuterium exchange mass spectrometry (H/
DXMS) to map the regions in MMP-1 involved in collagen
binding. Five MMP-1 regions showed delayed deuterium in-
corporation in the presence of collagen I (Fig. 3A and SI
Appendix, Fig. S3 and Table S1): Residues 145–161 (site 1) and
205–215 (site 3) in the Cat domain and residues 266–277 (site 4),
283–297 (site 5), and 330–346 (site 6) in the Hpx domain. Sites
4 and 5 were reported to reduce deuterium exchange upon
binding to a triple-helical peptide (22). Residues 179–193 (site 2)
showed increased deuterium incorporation in the presence of
collagen, suggesting that this region, which includes the loop
between the fifth β-strand and the second α-helix of the Cat
domain, is more exposed to the solvent in the collagen-bound
state. This loop contains a collagenase-characteristic cis-peptide
bond (23, 24), and it was identified as important for the colla-
genolytic activity of MMP-1 (23). Our data suggest that this loop
participates in collagenolysis dynamically. The combined H/
DXMS results indicate the general orientation of the collagen
triple helix binding to MMP-1 (Fig. 3, dashed line).
S10′ Exosite in the Hpx Domain. Considering the H/DXMS results
and the importance for MMP-1(E200A) binding of the two
leucines at the P1′ and P10′ positions of collagen, we searched
for hydrophobic sites in MMP-1 that could accept these residues.
For MMP-1 to cleave the Gly(P1)-Leu(P1′) bond, the P1′ Leu
must be located close to the S1′ pocket of the Cat domain. Using
this constraint, we docked a modeled triple-helical peptide to
MMP-1 and identified candidate residues in the Hpx domain
that might be involved in binding the P10′ Leu (Fig. 3A). They
were mutated and the mutant proteins tested for their collage-
nolytic activity (Fig. 3B). The most dramatic reduction of colla-
genase activity was observed with the F301Y single mutant and
the I271A/R272A double mutant, which exhibited 10% and 4%
of wild-type activity, respectively. Together, Phe301, Ile271, and
Arg272 in the Hpx domain participate in forming the hydro-
phobic S10′ binding pocket, or exosite. Ile271 and Arg272 were
identified in the H/DXMS experiment as part of site 4. Phe301 is
located between H/DXMS sites 5 and 6. It was not detected by
H/DXMS analysis most likely because its backbone amide is
buried in the Hpx domain.
Crystal Structure of an MMP-1(E200A)–Collagen Peptide Complex.
Based on the results of the collagen peptide binding studies,
we designed a triple-helical collagen peptide for cocrystallization
with MMP-1(E200A). The peptide spans positions P7-P17′ of
the collagen II cleavage site and is capped by two and three
nonnative Gly-Pro-Hyp triplets at the N and C termini, re-
spectively, to enhance triple helicity (Fig. 4A). The melting
temperature of this peptide was 31 °C (SI Appendix, Fig. S4). The
peptide formed a stable complex with MMP-1(E200A) in solu-
tion (SI Appendix, Fig. S5). Crystals of the complex were grown
I. (A) Binding of full-length MMP-1(E200A) and its in-
dividual Cat(E200A) and Hpx domains at 20 °C. (B) Binding
of MMP-1(E200A) at different temperatures. (C) Binding of
1 μM MMP-1(E200A) in the presence of the active-site in-
hibitor GM6001 at different temperatures.
Binding of MMP-1(E200A) to immobilized collagen
collagen II with proMMP-1(E200A), MMP-1(E200A),
Hpx domain, and Cat(E200A) domain. Error bars show
SDs from three repeats. Critical Leu and Ile residues at
P1′ and P10′ subsites are highlighted in the collagen II
portion of the peptide sequences surrounding the
collagenase cleavage site. ∼, bond cleaved by colla-
Screening of a triple-helical peptide library of
| www.pnas.org/cgi/doi/10.1073/pnas.1204991109Manka et al.
at room temperature and used to determine the structure at 3.0-
Å resolution (SI Appendix, Table S2).
The overall structure of human MMP-1(E200A) in complex
with collagen is similar to those reported for full-length pig (6)
and human (7) MMP-1 without collagen. The collagen peptide in
the complex with MMP-1(E200A) is an uninterrupted triple
helix ∼115 Å in length and bent by ∼10° near the Cat-Hpx
junction (Fig. 4B and SI Appendix, Fig. S6). The three chains of
the collagen triple helix are arranged with the characteristic one
residue stagger, resulting in a leading (L), middle (M), and trailing
(T) chain (25). All three chains contribute to MMP-1(E200A)
binding, creating an extensive interaction surface spanning nearly
60 Å in length. Complex formation buries 480 Å2of solvent-
accessible surface of the L chain, 440 Å2of the M chain, and
370 Å2of the T chain.
Interactions Between MMP-1(E200A) and the Collagen Peptide. The
majority of the interactions between the collagen peptide and the
Cat domain are of a polar nature (Fig. 4C and SI Appendix, Fig.
S7). Of the three collagen chains, the L chain is closest to the
active site of MMP-1, but the S1′ pocket remains empty. The
residue closest to the S1′ pocket is Gln(P4′L) (residues are
identified by their position relative to the scissile bond and
a subscript designating the collagen chain). The side chain of Gln
(P4′L) forms a hydrogen bond with the backbone amide of Tyr221
at the entrance of the S1′ pocket, and the backbone amide of Gln
(P4′L) forms a hydrogen bond with the side chain of Asn161. Leu
(P1′L) is 9 Å away from the S1′ pocket and stabilized by a hy-
drophobic contact with the zinc ligand His203. The Arg(P5′L)
side chain and backbone amide form hydrogen bonds with the side
chain of Tyr218 and the carbonyl oxygen of Pro219, respectively.
The M chain makes only a single contact with the Cat domain,
between the side chain of Gln(P4′M) and the side chain of Tyr218.
The T chain, however, makes extensive interactions with the upper
part of the substrate binding cleft, including five hydrogen bonds
(Fig. 4C and SI Appendix, Fig. S7). Some of these hydrogen
bonds are identical to those formed with a cysteine-switch sequence
(26–29) in proMMPs and with a predicted peptide substrate (30).
The interactions between the collagen peptide and the Hpx
domain are a combination of polar and apolar contacts, and are
mediated mainly by the M chain (Fig. 4D and SI Appendix, Fig.
S7). Consistent with our mutagenesis results, the side chain of
Leu(P10′M) is bound in a hydrophobic S10′ exosite pocket de-
lineated by Ile271, Met276, Phe301, Trp302, and the alkyl portion
of Arg272 (Fig. 4D). The S10′ pocket is surrounded by polar
residues (Arg272, Glu274, Arg285, and Gln335) that make a total
of five hydrogen bonds with the collagen peptide (SI Appendix,
Fig. S7). The interactions at the S10′ exosite are critical for col-
lagen recognition and collagenolysis by MMP-1 (Fig. 3B). In
addition, Ile(P7′M) forms hydrophobic interactions with Val300
and Phe301. The M and T chains make van der Waals contacts
with Phe289 and Tyr290, but these interactions evidently are
not critical, because the triple mutant MMP-1(F289A/Y290A/
P291A) retains 76% of wild-type collagenase activity (Fig. 3B).
Implications for Collagenolysis. The way in which the collagen
peptide is bound to the active-site cleft of MMP-1(E200A) is
more compatible with cleavage of the Gly(P3′L)-Gln(P4′L) bond
instead of the canonical Gly(P1)-Leu(P1′) bond (31). To test
whether MMP-1 can cleave also before Gln(P4′), we performed
N-terminal sequencing of the products generated by active
MMP-1 at 25 °C and 37 °C. We found that only the Gly(P1)-Leu
(P1′) bond was cleaved in the peptide, as in native collagen II,
and concluded that the mode of collagen binding in the crys-
tallized complex is unproductive. A productive mode could be
achieved by a substantial movement of the Cat domain relative
to the Hpx domain, but this necessitates the breaking of the Cat-
Hpx interface (19). We favor an alternative mechanism that
leaves the interface intact. The helical symmetry of homotrimeric
collagen dictates that axial rotation of the collagen homotrimer
(in other words, binding of MMP-1 to a different set of collagen
chains) changes the register of the chain passing through the
active site cleft, but leaves all other interactions unchanged (Fig.
5 and SI Appendix, Fig. S8). If Leu(P10′L) instead of Leu(P10′M)
is made to occupy the hydrophobic S10′ exosite, the T chain
becomes the chain that interacts most closely with the active site,
and now Leu(P1′T) is situated near the S1′ pocket. Notably, this
productive binding mode does not alter any of the extensive
interactions with the upper part of the substrate binding cleft
(now made by the M chain) or with the S10′ exosite in the Hpx
domain (now made by the L chain) (Fig. 5 and SI Appendix, Fig.
S8). The energy difference between the observed unproductive
and putative productive complex is likely to be small, and the two
forms may coexist in solution. The unproductive complex may be
favored by the E200A mutant of MMP-1 or it may have crys-
tallized preferentially in our experiment.
Starting from the modeled productive complex (SI Appendix,
Fig. S8), it proved straightforward to achieve the first transition
state of collagenolysis, in which one collagen chain is inserted
into the active site (Fig. 6). By stretching the P1′-P6′ region of
the T chain and reorienting three residues preceding the scissile
bond, it was possible to insert Leu(P1′T) into the S1′ pocket in
a manner that fully agrees with the observed binding mode of
single-chain peptidomimetic MMP inhibitors (8, 32). The Cα
atom of Leu(P1′T) moves by less than 4 Å during this localized
excursion from the regular triple-helical structure, and only four
collagen interchain hydrogen bonds are broken in the transition
state. We propose that the energy penalty for this local unfolding
event is compensated by the favorable interactions that the en-
zyme forms with the looped-out collagen chain.
Our study has revealed an unprecedented proteinase–substrate
interaction. The most striking feature of the MMP-1–collagen
complex is the extensive interaction of all three collagen chains
with the two domains of MMP-1, which explains the strict re-
quirement of the Hpx domain for efficient collagenolysis (3). The
individual Cat or Hpx domains of MMP-1 showed very little
collagen binding in our experiments, but when linked together,
they acted cooperatively in a temperature-dependent manner.
The affinity for collagen I increased up to 37 °C, a temperature
that would not cause collagen to denature within the time frame
of the experiment (∼2 h) (33). Above this temperature the
binding sharply decreased. This temperature dependence, which
is also unusually pronounced in the rate of collagen cleavage by
mutagenesis. (A) Sites 1 and 3–6 are protected from deuterium incorporation,
and site 2 shows enhanced deuterium incorporation upon collagen binding.
The sites are mapped onto the crystal structure of MMP-1(E200A) (7). Resi-
dues potentially involved in collagenolysis that were mutated are indicated.
Dashed line, predicted collagen binding direction. (B) Relative initial velocities
of collagen I cleavage by MMP-1 and its variants normalized to WT.
Collagen I footprint on MMP-1(E200A) determined by H/DXMS and
Manka et al.PNAS
| July 31, 2012
| vol. 109
| no. 31
MMP-1 (34), can be explained by the relative thermoflexibility of
the triple helix at the collagenase cleavage site (35–38) and the
contribution of interdomain flexibility of MMP-1. The co-
operative binding is therefore energetically most favored close to
body temperature, when the triple helix is locally looser but the
overall fold of the substrate is still maintained. A complete loss
of triple helicity makes collagen bind less tightly to MMP-1, as
evidenced from a Kmvalue of ∼1 μM for collagen I compared
with 4–7 μM for gelatin (39). The small active-site inhibitor
GM6001 abolished the temperature-dependent increase of col-
lagen binding, indicating that the catalytic site of MMP-1 is not
only involved in the final hydrolysis step, but also in the unfolding
machinery, which uses the active site to extract one collagen
chain to form the first transition state of collagenolysis (Fig. 6).
Apart from providing a favorable environment for the loose
triple helix at the cleavage site, what other mechanism(s) might
contribute to collagenolysis? Comparison of the crystal structure
of proMMP-1 (29) with the structure of the activated form of
MMP-1(E200A) (6, 7) suggested that the Cat-Hpx linker region
confers interdomain flexibility. Such flexing was further demon-
strated for MMP-1 by NMR and small angle X-ray scattering
studies (40, 41) and seems to be a common property of MMPs
(42, 43). Our crystal structure of MMP-1 bound to collagen has
a more closed conformation than free MMP-1 (SI Appendix, Fig.
S9). This change in interdomain angle is accompanied by the
bending of the collagen peptide at a point halfway between the
Cat and Hpx domains. Thus, domain motions in MMP-1 may
facilitate structural changes in the triple helix. Our structural
results show how the two domains of MMP-1 anchor two of the
collagen chains (M and L in the modeled productive complex)
and position the third (T) chain near the active site. Stretching of
the T chain by interdomain flexing of the enzyme may assist in
local collagen unfolding. Because the P1′ residue of the trailing
chain is poised above the S1′ pocket, transient excursions of the
triple helical collagen peptide complex. (A) Se-
quence of the collagen peptide used for coc-
within 4 Å distance of the enzyme in the com-
plex are colored. The subsite designation is in-
dicated for the leading chain. (B) Stereoview of
the MMP-1(E200A)–collagen peptide complex.
The collagen chains are colored cyan (L), green
(M), and red (T), and the enzyme is shown as
a gray surface with areas within 4 Å distance of
the L, M, and T chains colored correspondingly.
Magenta sphere, active-site zinc ion. (C) Ster-
eoview of the interactions between collagen
chains (colored as in B) and the active site cleft
of MMP-1(E200A). Dashed lines indicate hydro-
gen bonds. (D) Stereoview of the interactions of
the collagen chains with the Hpx domain. Se-
lected residues making enzyme-substrate con-
tacts are labeled with respective colors and
shown in stick representation (N, dark blue;
Crystal structure of the MMP-1(E200A)–
| www.pnas.org/cgi/doi/10.1073/pnas.1204991109Manka et al.
scissile bond can be efficiently captured by the enzyme, leading
to the stabilization of the first transition state of collagenolysis
(Fig. 6). Although thermal relaxation of the cleavage site is im-
portant for MMP-1 binding, this alone is not sufficient for effi-
cient collagenolysis (17, 37). We propose that mammalian
collagenases do not serve merely as passive acceptors of spon-
taneously occurring unfolded collagen states (44, 45), but they
promote a local perturbation of the triple helix that facilitates
collagenolysis. The recently reported NMR study supports this
notion by demonstrating a weakening of the collagen interchain
contacts in the complex with MMP-1 (19).
Our mechanism of collagenolysis (Fig. 6) differs from that
derived from NMR experiments (19) in that it does not require
a separation of the Cat and Hpx domains. Bertini et al. (19), who
also used the E200A mutant of MMP-1, found that the Cat
domain interacted mainly with the L chain and the Hpx domain
mainly with the L and M chains. These results agree with our
crystal structure of an unproductive complex. In their modeling,
Bertini et al. (19) imposed a productive interaction of the
L chain with the S1′ pocket, which forced the breaking of the
Cat-Hpx interface. Our interpretation requires only small con-
formational changes in the enzyme but postulates the co-
existence of productive and unproductive binding modes.
The crystal structure of a bacterial collagenase comprising of a N-
terminal activator domain, a glycine-rich linker, and a peptidase
domain has been reported (46). Like MMPs, this bacterial colla-
genase requires an ancillary activator domain (unrelated to the Hpx
domain of MMPs) to cleave triple-helical collagen. The authors
proposed that the enzyme clamps collagen between the activator
and peptidase domains of the saddle-shaped structure. Our MMP-
collagen structure shows that MMP-1 binds collagen in an extended
manner, rather than surrounding it. These differences notwith-
standing, interdomain plasticity seems to be a common feature of
enzymes that can cleave the intractable collagen triple helix.
Most MMPs are believed to have specific substrate recognition
sites distant from the catalytic site, which are referred to as
exosites. Apart from the Hpx domains of collagenases, examples
include the fibronectin type II repeats in MMP-2 and MMP-9,
which contribute to the binding and degradation of elastin, gel-
atin, and type IV collagen (47). Recent NMR and mutagenesis
studies of the Cat domain of MMP-12 characterized several
exosites for elastin (48, 49). Similar studies were carried out with
the isolated Hpx domain of MMP-1 and a triple-helical collagen
peptide (40) and suggested a role for Phe282 (Phe301 in the
numbering used in ref. 40) in binding of collagen. However, the
exact modes of interaction between the enzyme and the target
substrate have not been elucidated in these studies. Our crystal
structure has revealed in detail the interactions that occur be-
tween full-length MMP-1 and a triple-helical peptide long
enough to define all relevant contacts. Phe282 is buried in the
Cat-Hpx interface in our structure and not involved in collagen
binding. Defining the exosites in proteolytic enzymes has im-
portant implications for the development of specific inhibitors.
The hydrophobic S10′ exosite in MMP-1 is a particularly at-
tractive target because it is essential for collagenolysis. Recent
studies of Robichaud et al. (50) showed the importance of Leu at
the P10′ subsite of collagen III for the cleavage by MMP-1.
Noncollagenous extracellular matrix and nonmatrix substrates
are cleaved by the Cat domain of MMP-1 (16). Therefore, typical
inhibitors targeting the S1′ pocket block all MMP-1 activities,
whereas inhibitors directed against the S10′ exosite are predicted
to selectively inhibit collagenolysis. This exosite of MMP-1 is
largely conserved in other MMPs, but to our knowledge it is
specific for collagenolytic activity. We propose that molecules
specifically binding the S10′ exosite be therapeutically useful in
diseases associated with accelerated collagen breakdown such as
arthritis, cancer, aneurysm, and atherosclerosis.
Materials and Methods
Proteins and Peptides, Binding Assays, H/DXMS Analysis, and Enzyme Activity
Assay. Recombinant proMMP-1(E200A), its Cat(E200A) and Hpx domains, and
MMP-1 variants were produced in Escherichia coli. Collagen I was purified
from Guinea pig skin and treated with pepsin. The library of triple-helical
peptides of human collagen II (Toolkit II) and all other collagen peptides
were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Col-
lagen and peptide binding assays were carried out in ELISA format by using
biotinylated proteins and detected by streptavidin-horseradish peroxidase
system. Local hydrogen/deuterium exchange kinetics of MMP-1(E200A) free
and bound to collagen I were analyzed by digestion with pepsin and mass
spectrometryand quantifiedby usingHX-Express software.The
sequence alignment on the left, the trailing chain is shown twice to emphasize the circular nature of the chain arrangement. The P1′ and P10′ residues are
boxed, and residues interacting with MMP-1 are in bold. The structural models on the right show that the unproductive and productive binding modes are
related by a simple rotation/translation of the collagen triple helix. Note that only the interactions with one collagen chain are changed (L in the unproductive
mode, T in the productive mode); the interactions with the other two chains are the same in both modes.
Modes of collagen binding to MMP-1. Shown is relationship between the unproductive and productive collagen binding modes (see text). In the
productive complex. The upper rim of the catalytic site cleft anchors the
M chain (green), and the Hpx domain anchors the L chain (cyan). These
interactions position the T chain above the active site. Interdomain flexing of
the enzyme bends the collagen triple helix and facilitates the insertion of
Leu(P1′) of the T chain into the S1′ pocket.
Model of the first transition state of collagenolysis based on the
Manka et al.PNAS
| July 31, 2012
| vol. 109
| no. 31
collagenolytic activity of MMP-1 variants was determined by SDS/PAGE and
densitometric analyses of collagen I cleavage products. Detailed methods
are described in SI Appendix.
Crystallization, Data Collection, and Structure Determination. MMP-1(E200A)
purified by size exclusion chromatography. Crystals of the complex were
obtained at pH 8.5 and diffracted to 3.0 Å resolution. The structure was solved
by molecular replacement and refined to a free R-factor of 0.273. Detailed
methods are described in SI Appendix. Data processing and refinement statistics
are listed in SI Appendix, Table S2. The coordinates of the MMP-1(E200A)–
collagen complex have been deposited in the Protein Data Bank (ID code 4AUO).
ACKNOWLEDGMENTS. We thank Noriko Ito for construction of MMP-1
mutants, Ida B. Thøgersen for peptide sequencing, the staff at Diamond
beamline I24 for help with X-ray data collection, and Peter Brick for many
helpful discussions. Protein visualization and modeling was done in PyMOL
(www.pymol.org). The work was supported by Wellcome Trust Grants
068724, 075473, 094470, and 083942; the Medical Research Council, Arthritis
Research UK; and Cancer Research UK. E.H. is a Wellcome Trust Senior
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| www.pnas.org/cgi/doi/10.1073/pnas.1204991109Manka et al.