From the Department of Structural Research, Max
Planck Institute for Biochemistry, D-82152 Martinsried, Germany,
Pharmaceutical Research, Roche Diagnostics GmbH, D-82372
Penzberg, Germany, and ¶ Molecular Design, Pharmaceuticals
Division, F. Hoffmann La Roche Ltd., CH-4070 Basel, Switzerland
Received for publication, August 6, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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The individual zinc endoproteinases
of the tissue degrading matrix metalloproteinase (MMP) family share a
common catalytic architecture but are differentiated with respect to
substrate specificity, localization, and activation. Variation in
domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four
distinct classes (Nagase, H., and Woessner, J. F. J. (1999) J. Biol. Chem. 274, 21491-21494). Exploitation of
these differences may be decisive for the design of anticancer or other
drugs, which should be highly selective for their particular MMP
targets. Based on the 1.8-Å crystal structure of human neutrophil
collagenase (MMP-8) in complex with an active site-directed inhibitor
(RO200-1770), we identify and describe new structural determinants for
substrate and inhibitor recognition in addition to the primary
substrate recognition sites. RO200-1770 induces a major rearrangement
at a position relevant to substrate recognition near the MMP-8 active site (Ala206-Asn218). In stromelysin (MMP-3),
competing stabilizing interactions at the analogous segment hinder a
similar rearrangement, consistent with kinetic profiling of several
MMPs. Despite the apparent dissimilarity of the inhibitors, the central
2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor
RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor
batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T.,
Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995)
Eur. J. Biochem. 228, 830-841) for binding to MMP-8.
The two additional phenyl and piperidyl ring substituents of the
inhibitor bind into the S1' and S2' pockets of MMP-8, respectively. The
crystal lattice contains a hydrogen bond between the O The matrix metalloproteinases
(MMPs),1 one of the five
families that form the metzincin group of zinc proteinases (3),
function to degrade the extracellular matrix during embryonic
development, reproduction, and tissue remodeling (1) but are
disregulated in arthritis, cancer, and other diseases. The
"minimal" MMPs matrilysin and endometase (MMP-7 and MMP-26,
respectively), have a Zn2+ and Ca2+ binding
catalytic domain, and an N-terminal pro-domain. All other known MMPs
possess additionally a hemopexin-like domain near the C terminus, and
further domain insertions differentiate MMP subfamilies. Gelatinases A
and B (MMP-2 and MMP-9) possess three fibronectin type II-like repeats
inserted at a loop in the catalytic domain; these form an independent
folding domain adjacent to the catalytic domain. Membrane-type MMPs
possess an anchoring transmembrane helix C-terminal to the
hemopexin-like domain (4). Hierarchical regulation of MMP activity
occurs on many levels, including gene expression control (1, 5),
proteolytic activation of MMP zymogens (6), inhibition by endogenous
tissue inhibitors of metalloproteinases (7), and both positive and
negative proteolytic feedback loops (8, 9). Crystal structures of
several MMPs have been determined (for a review, see, e.g.,
Ref. 10), revealing overall domain structures, catalytic mechanisms,
and many aspects of MMP regulation mechanisms; these include
collagenase 1 (MMP-1) (11, 12) and collagenase 2 (MMP-8). Structures of
the latter are represented by two forms of the catalytic domain,
resulting from activation cleavage alternately at two cleavage sites,
leaving either Met80 (13, 14) or Phe79 as the
N-terminal residue (15). The latter form is "superactivated," as
Phe79 forms a salt bridge with Asp232 and
thereby prevents the N-terminal sequence from transient or other
interference with the active site. The result is a 3-fold increase in
activity compared with activation cleavage at Met80
(16).
As their early nomenclature implies, collagenases I, -II, and -III (17)
(MMP-1, -8, and -13, respectively) degrade mainly fibrillar collagens
(18-20), although the structural origin of this specificity is not
well understood (4). Disruption of MMP tissue remodeling function
causes a variety of disorders, including cancer (tumor growth, invasion
and metastasis), rheumatoid arthritis and osteoarthritis, and a variety
of diseases involving neovascularization. The resulting clinical need
has fostered an enormous interest in the development of inhibitors
against MMPs. As part of these efforts, crystal structures of MMPs with
a variety of synthetic inhibitors have been determined. For MMP-8,
complexes reported include peptide mimetics, hydroxamic acid
derivatives (2, 14, 21, 22), phosphinamides and sulfodiimines (23, 24),
thiadiazole (25), and malonic acid derivatives (26, 27).
Here we describe the 1.8-Å crystal structure of MMP-8 inhibited by a
barbituric acid derivative. Conformational rearrangements accompanying
the inhibitor binding lead to a new and highly ordered crystal packing
arrangement. The high resolution structural data enables a thorough
analysis of determinants of MMP selectivity toward both low molecular
weight substances as well as substrate classes. A previously unreported
cis-peptide bond (Asn188 Materials--
MMP-1, MMP-3, and MMP-8 were kindly provided by
Profs. G. Murphy (University of East Anglia, Norwich, United Kingdom),
H. Nagase (Imperial College, London, United Kingdom), and H. Tschesche (University of Bielefeld, Bielefeld, Germany), respectively; MMP-2 and
MMP-9 were obtained from Roche Molecular Biochemicals (Penzberg, Germany); MT1-MMP and MT3-MMP were provided by Prof. J. Foidart (Université de Liège, Liège, Belgium). The inhibitors
RO200-1770, RO204-1924, I-COL043, RO206-0027, and RO206-0032 were
synthesized as described previously (28). The fluorogenic substrate
M-1855 (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2)
was purchased from Bachem (Heidelberg, Germany); all other chemicals
were of highest purity commercially available.
Inhibition Assay--
All measurements were performed at
25 °C using a buffering solution of 50 mM Tris, pH 7.6, 100 mM NaCl, 10 mM CaCl2. Based on
multiple measurements, all data are precise to within 5%. Depending on
activity, enzymes were used at 5-50 nM concentration range with a substrate concentration of 2.55 µM. The enzyme was
briefly pre-incubated with the inhibitor at a resultant
Me2SO concentration of 1%. The reaction was started with
the addition of the substrate M-1855. Substrate was excited at 280 nm
and the substrate fluorescence was monitored at 346 nm using the
FuoroMax-3 fluorometer (SPEX, Horiba Group, Grasbrunn/Munich, Germany).
Crystallization, Data Collection, and Structure
Refinement--
MMP-8 was concentrated to 8 mg/ml and then mixed with
3-fold molar excess of an aqueous solution of RO200-1770 for a final MMP-8 concentration of 6 mg/ml. 3 µl of protein-inhibitor complex was
mixed with 2 µl precipitant solution containing 100 mM
cacodylate pH 5.5-6.5, 10 mM CaCl2, 100 mM NaCl, and 10% polyethylene glycol 6000. The hanging
drop was equilibrated by vapor diffusion at room temperature against a
reservoir containing 1.0-1.5 M phosphate buffer. Data were
collected on a multiwire detector (X1000, Bruker AXS) to 1.8-Å
resolution and processed using SAINT data reduction software (29),
yielding an agreement of redundant measurements of
Rmerge = 9.3% over all data and 41% in the
outer resolution shell (completeness 98% and 87%, respectively). The
space group of the crystal was determined as I222 with unit
cell dimensions a = 61.02 Å, b = 69.24 Å, c = 88.47 Å. The orientation and translation of
the molecule within the crystallographic unit cell was determined with
Patterson search techniques (30-32) using the program AMoRe (33).
Electron density calculation and model building proceeded using the
program MAIN (34). The structure has been refined by using the program
X-PLOR (35) to a crystallographic R-value of 21.1%
(Rfree = 29.6%) with bond deviations of 0.009 Å and angle deviations of 1.7° from ideality (36). The molecular
structure was analyzed and compared using appropriate tools within the
program MAIN (34).
Inhibitor Conformation--
The inhibitor RO200-1770 is a
barbituric acid derivative, doubly substituted with phenyl and
4-ethanolpiperidyl rings as depicted in Fig.
1. The barbiturate ring chelates the zinc
and rigidly orients the two cyclic substituents into the S1' and S2'
substrate binding sites. Neither substituent ring system appears
strained by the protein environment, although their relative
orientations may be induced by protein binding. The phenyl moiety
occupies the MMP-8 binding site and is perfectly planar to within the
1.8-Å resolution. The electron density observed for the piperidine
ring allows an interpretation whereby two chair conformations related by a 180° rotation along the C5-pN1 bond
might be superimposed; either conformation would allow favorable
hydrophobic contacts in the S2'-site. Adopting an all-trans
conformation, the alcohol group points toward the solvent.
The relative orientations of the rings of the inhibitor may be
described by considering the ring planes and the bonds linking the
substituent rings to the C5 atom of the barbiturate ring. The C5-fC1 bond linking the phenyl ring is
nearly perpendicular to the plane of the barbiturate ring (excluding
C5). This arrangement necessarily orients the plane of the
phenyl ring likewise perpendicular to the barbiturate plane. The
dihedral angle
C6-C5-fC1-fC2 fixes the ring orientation with an eclipsed geometry (at 1.2°, while the
C4-C5-C1-fC2
dihedral is staggered at 60.8°). In contrast, the
C5-pN1 bond lies nearly in the plane of the
barbiturate, extending the P2'-piperidyl ring away from the
barbiturate; all dihedrals across the C5 Protein-Inhibitor Interaction--
The MMP-8 substrate recognition
sites are shown schematically in Fig. 3A. Comparison with
the binding mode of RO200-1770 as depicted in Fig. 3B
highlights the inhibitor binding at the "primed" substrate
recognition sites and at the Zn2+ ion. The Zn2+
is coordinated by atoms N3 and O2 of the
barbiturate ring. The Zn2+
The pentacoordinated Zn2+ binding geometry resembles a
highly distorted trigonal bipyramidal structure with O2,
N
In contrast to the polar interactions of the barbiturate ring, the
interactions mediated by the phenyl and piperidyl rings are
predominantly hydrophobic and involve the S1' and S2' pockets, respectively. The most prominent interaction in the S1' pocket is the
ideally parallel planar stacking of the phenyl ring and His197 at a distance of 3.6 Å (Fig.
4). The conserved Leu160
contributes to ligand binding also with its side chain in the S1' site.
The phenyl ring does not by itself fill the S1' site, but leaves space
filled by a network of three ordered water molecules. The first of
these (Sol595) is probably incompletely occupied and forms
hydrogen bonds with the inhibitor, with MMP-8, and with a second water
molecule. The proximity of the inhibitor phenyl fC4 atom to
Sol595 (3.1 Å) indicates a O ... H-C interaction
(38). The carbonyl group of Leu193 forms a 2.9-Å hydrogen
bond with Sol595 with, however, an unfavorable
C=O193-O595 angle of 113°. The second water
molecule, Sol602, is positioned deeper inside the S1'
pocket at a hydrogen bonding distance of 2.7 Å from
Sol595. Sol602 in turn is hydrogen-bonded (2.8 Å) with the third solvent molecule in the S1' pocket,
Sol592. Sol592 is in a channel bounded by
Arg222, which forms a link between the three water network
in S1' and, via Sol667 (2.9 Å from Sol602),
water in the adjacent cavity. Mutation of Arg222 would
connect the two cavities, opening a "back door" to S1' for solvent
access. The guanidinium group atoms N
The hydrophobic interactions of the piperidine ring are mediated by
aliphatic surfaces from
Pro217-Asn218-Tyr219 at the
"southern" rim of S2' and by the main chain
Gly158-Ile159-Leu160 at the
"northern" rim. The latter residue (Leu160) separates
the S1' and S2' pockets. No ordered water molecule can be detected in
the vicinity of the hydroxyl group pOH9, although the
position of this solvent exposed ethanol group is well defined by
electron density (Fig. 2) and is thus presumably hydrated by disordered water.
Protein Conformational Changes--
Significant differences are
apparent in the protein structure compared with previously determined
MMP-8 structures (2, 14, 21). The catalytic Zn2+ ion of the
three reference structures occupies the same position to within 0.2 Å;
it is, however, shifted from that average position by 0.6 Å in the
RO200-1770 complex structure. Corresponding shifts of the
Zn2+ protein ligand positions are also apparent, with the
respective N
Of the two partial sequences harboring the Zn2+ binding
histidine residues, the loop
Ala206-His207-Asn218 is more
exposed to the solvent and anchored by fewer protein contacts than the
internal helix
L191-H197EXXH201-L203.
The conformation of this loop is altered by several effects associated
with the binding of RO200-1770. First, the greater inherent plasticity
of this loop leads to greater compensation by the Zn ligand His207 for
shear stresses induced at the catalytic site. Second, the
Pro217-Asn218 peptide bond is rotated by
~100°, evidently to prevent a repulsive interaction between the
barbiturate C4=O4 keto group with the Pro217 carbonyl. Third, residues Ser209,
Tyr216, Pro217, and Asn218 form
crystal contacts. These effects in combination lead to a translation
along the entire loop from Ala206 to Pro217,
which, however, is relatively rigid, leaving most dihedral angles similar to those in the reference structures. In the "north" rim of
the active site, the largest change compared with the inhibitor free
MMP-8 structure is a 0.98 Å displacement and disorder of the
Ile159 side chain; the electron density shows a branched
but symmetric side chain interpretable as two equally populated
rotamers, which "swap" C Enzyme Inhibition Analysis--
Utilizing the crystal structure of
the MMP8-RO200-1770 complex, several follow-up compounds were
synthesized and tested against the panel of metalloenzymes shown in
Table II. The lead compound RO200-1770
shows broadly nonspecific micromolar inhibition, excepting only
stromelysin 1 (MMP-3) with its ~10-fold weaker binding affinity to
RO200-1770. To facilitate synthesis, the piperidine of the lead
compound RO200-1770 was substituted by an essentially isosteric piperazine, RO204-1924. The almost uniform decrease in binding affinity
might be rationalized by higher desolvation penalties for piperazine
binding. The theoretical clogP values calculated for
1,4-dimethylpiperidine (1.9) and 1,4-dimethylpiperazine (0.8) support
this hypothesis (39). I-COL043 and RO206-0027 represent the results of
two orthogonal approaches to optimize P1'-S1' and P2'-S2' binding,
respectively. For each inhibitor, an ~10-fold increase in inhibition
toward MMP-8, -2, -9, and -3 was accomplished, while inhibition of
MT1-MMP and MMP-1 was weakened or remained relatively unchanged. With
its 4-fold weaker inhibition of MMP-1, I-COL043 showed significantly
enhanced selectivity potential against the latter enzyme. The P1' and
P2' optimizations of I-COL043 and RO206-0027 are combined in RO206-0032
and the inhibition values demonstrate, to a first approximation,
additivity of the effects for MMP-8, -2, -9, and MT1-MMP. The
improvement in its binding affinity to stromelysin (MMP-3) is less
distinct, while fibroblast collagenase (MMP-1) binding averages rather
than sums the effects of the precedent compounds.
Crystal Packing Effects--
The MMP8-RO200-1770 complex did not
crystallize as previously described (26), but also under the previously
reported crystallization conditions formed the crystal packing
arrangement described here. Thus, the inhibitor induces a
conformational rearrangement that leads to the new crystal packing. As
discussed above, repulsion between the barbiturate
C4=O4 and Pro217 carbonyl groups
displaces the Pro217-Asn218 peptide. Its new
orientation is stabilized by hydrogen bonds to the alcohol of
Ser209 of a neighboring molecule in the crystal. This
serine alcohol also forms a hydrogen bond (2.7 Å) with the
N Secondary Substrate Recognition Site: A Collagenase Type I
Characteristic Cis-peptide Bond--
The recognition and processing of
natural collagen substrates is known to involve the C-terminal
hemopexin-like domain in addition to the active site (4, 40, 41). The
relative domain arrangement of the catalytic and C-terminal domains, as
seen for MMP-1 (11) and MMP-2 (42), shows the importance of the primed substrate recognition sites, since these are located at the interface of the two collagenase domains. Intriguingly,
Asn188-Tyr189, located at the corridor
connecting the catalytic and the C-terminal domain, adopts a
cis-peptide bond (Fig. 5). Although not
yet recognized, this cis-peptide bond is not unique to the present
crystal form; re-inspection confirmed its presence also in the
alternative crystal form (26). This cis-peptide bond is located on the
solvent-exposed loop preceding the "catalytic" Inhibitor Conformation and Its Interaction with the
Protein--
Although identified as potent collagenase inhibitor by an
independent screening program, the barbiturate-based inhibitor family exhibits striking similarities with well characterized classes of
inhibitors, namely hydroxamic and malonic acid-based compounds (2, 26).
Fig. 3C illustrates that the Zn2+ chelation
geometry of the hydroxamate, exemplified by batimastat, is mirrored by
the barbiturate with its N3 nitrogen substituting for the
keto group of the hydroxamate. Additionally, the interaction of the
barbiturate N1-H1 and O6 with the
protein backbone Ala160-Ala161 parallels that
of batimastat (Fig. 3). A subtle difference is found at the
O6 interaction of the barbiturate ring, since the additional amide interaction with Ala161 could stabilize a
greater negative charge on O6.
These findings present opportunities with challenges. The structural
similarity of both inhibitor classes for example enables the
application of knowledge of optimization criteria for one class to the
other. On the other hand, the similarity might also indicate a
limitation in finding specific metalloproteinase inhibitors; the
presence of a similar metal chelation topology in independently identified and structurally unrelated lead compounds indicates that the
Zn2+ binding follows a rather universal recognition motif,
which dominates the binding characteristics. Consequently, many if not
most potent active site directed Zn2+ protease inhibitor
will exploit such a universal binding motif and are likely to exhibit a
low specificity profile, at least prior to optimization.
The Barbituric Acid Carries No Net Charge--
The charge
assignment of the Zn2+-chelating barbiturate ring aids an
understanding of the binding interaction. For the crystallization experiment, the pH was adjusted to 6.0 (see "Experimental
Procedures.") This information is, however, insufficient to allow for
a reliable prediction of the protonation state of the barbiturate ring.
First, its pKa varies dramatically with the presence
of ring substituents. Whereas the pKa of
unsubstituted barbituric acid is about 4, its 5,5-diethyl substituted
analog ("barbital") has a pKa of around 8 (37).
Second, the surrounding protein will also strongly affect the
protonation of the barbiturate.
To address this issue, we inspected each polar group of the inhibitor
for possible hydrogen bonding partners. The 2.0-Å distance of the
catalytic Zn2+ to N3 excludes its protonation,
and the O2H2 hydroxyl group is necessary to
avoid repulsion of the carboxylate of Glu198.
N1 and O6 are involved in main chain hydrogen
bonds. Consequently, their protonation appears well defined as depicted
in Fig. 1. O4 is the only polar group without apparent
attractive interactions with the protein. However, the reorientation of
the Pro217 carbonyl described above would seemingly not
occur if O4 is protonated as an alcohol. These arguments
summarize the case for the formula depicted in Fig. 1, which carries no
net charge. Tunneling of the proton H2 (Fig. 1), which
bridges the carboxylate group of Glu198, however, transfers
a partial negative charge to O2 and by resonance also to
N3 (Fig. 1). (A second line of investigation using
conformational correlation analysis of the 1.8-Å resolution structure
presented here with barbiturate derivatives deposited in the Cambridge
small molecule data base was not conclusive.)
S1' and S2' Interaction, and Enzyme Inhibition
Profiles--
Compared with MMP-8, human fibroblast collagenase
(MMP-1) has a more restricted S1' site with its Arg instead of Leu at
position 193. Its guanidinium group approximately occupies the three
S1' solvent sites of MMP-8, namely Sol595,
Sol602, and Sol592. Conversely, three solvent
molecules are found near Thr222 in MMP-1, where in MMP-8
the guanidinium group of Arg222 is found. It appears,
therefore, possible to enlarge the MMP-1 S1' subsite to an MMP-8 size
by swapping its Arg side chain and solvent molecules. Although such a
swapped conformation has been confirmed (12) for MMP-1, the rehydration
is likely to create a considerable kinetic barrier. Consequently, MMP-1
is expected to bind large P1' residues with a
kon kinetic rate considerably lower than for
MMP-8. The S1' site of TACE appears rather too large to properly
accommodate the large P1' residue of ICOL 043 and RO206-0032 (Table
II). A unique feature of the TACE active site (44) is the occurrence of
Ala at the equivalent position of the strictly conserved
Tyr219 (MMP-8 numbering) of MMPs. This renders the TACE S1'
site both larger and less hydrophobic than in MMPs by almost completely removing the barrier to the S3' site. Consequently, the hydrophobic P1'
residue of ICOL 043 and RO206-0032 is not optimally anchored in the
TACE S1' pocket. Further, incomplete dehydration of the voluminous site
is likely to disrupt the solvent structure within the TACE S1' site
(44) without the energy compensation of a good fit.
Considering MMP-3, the southern rim of the active site, and in
particular Pro221 (Pro217 in MMP-8), is
rigidified by His224 (Ala220 in MMP-8), which
hydrogen-bonds via its N Optimization of Binding Affinity and Selectivity--
Excepting
C4=O4, all polar groups of the barbiturate bind within the protein
matrix and should, therefore, remain invariant in optimization
strategies. The C4=O4 keto group, however, presumably weakens the
binding because of repulsive interactions with the Pro217
carbonyl group, as described in the paragraph "Protein Conformational Changes." Consequently, we identify this group as a major variable in
optimizing the Zn2+-chelating moiety. In fact, modeling
studies together with small molecule crystallographic data base
analyses indicate that analogous five-membered ring systems might be an
appropriate substitute Zn2+-binding group, provided that
the Zn2+ chelating properties are maintained. For example,
deletion of the C4=O4 ketone and retention of
the position of the chelating group
N3-C2-OH2 will allow the new
five-membered ring to relax to a chemically reasonable geometry. With
appropriate restraints for the relaxation, the result will still
possess favorable hydrogen bonding interactions between the
HN1-C6=O6 segment and the protein. To compensate for concomitant displacements of the phenyl ring, it
would be necessary to add a spacer atom to restore unstrained occupancies of both the S1' and S2' pockets.
Additional optimization approaches are suggested by kinetic analyses of
earlier x-ray structures of MMPs with peptidic, hydroxamic, and malonic
acid-based inhibitors (2, 26, 27, 45), whereby significant improvement
in binding affinity is achieved by better filling the respective
binding pockets (see Ref. 46 for a comprehensive review of these
approaches). Expansion of the P1' residue with an appropriate
heterocyclic ring should supply both the necessary flexibility to
optimally fill the curved S1' pocket and the hydrophilicity to
adequately replace the binding sites of the three water molecules found
in the S1' pocket. In summary, the major contribution to specificity
can be attributed to the P1'-S1' interaction, while both substituents
similarly contribute to MMP-8 binding (Table II).
Protein Conformational Changes--
As described under
"Results," the most striking structural changes induced by
inhibitor binding occur near the catalytic Zn2+ with a
major contribution from repulsive interactions between the
C4=O4 ketone and the Pro217
carbonyl group. For the collagenases, considerable flexibility near the
active site environment appears physiologically necessary for triple
helical peptide processing (47). The flexibility observed in the
present structure suggests that the loop
Ala206
The intermolecular crystal contact
Ser209 Type I Collagen Recognition Exosite--
Independent
investigations by others on rat MMP-8 have shown the 188-loop to be
required for collagenase activity. The single site-directed mutation to
N209K, corresponding to N188K in human MMP-8, disrupts collagenolytic
activity.2 In addition,
hybrid molecule studies involving stromelysin 1 (N-terminal) and
collagenase 1 (C-terminal) underscore the importance of this loop for
collagenolytic activity. The segment
R181WTNNFREY189 of collagenase 1 is
critical for triple-helicase activity (49). In addition to collagenase
1, 2, and 3, the two gelatinases MMP-2 and MMP-9 have tyrosine at
position 189, both of which are preceded by a large insertion of three
fibronectin II domains (Fig. 6). These
domains also are known to be critical for substrate recognition (4, 48,
50). Therefore, the 188 exosite serves as a collagen substrate
recognition site in both collagenases (MMP-1, -8, and -13) and
gelatinases (MMP-2 and -9). This proposed substrate recognition site is
the position of the cis peptide bond described here for MMP-8 and
predicted for MMP-1. As such, it distinguishes collagenases 1 and 2 (MMP-1 and -8) from the other MMPs known to cleave collagen that have a
glycine at this position, including MMP-13 (17), MMP-14 (51), and
MMP-18 (52). Thus, collagenase 3 (MMP-13) (12, 53), MMP-14 (54), and
presumably MMP-18 have a different backbone conformation in this loop
segment. This structural relationship is reflected by the biochemical
properties of the respective enzymes. MMP-13 is distinct from MMP-1 and
-8, as it preferentially hydrolyzes type II collagen, whereas the
enzyme was 5 or 6 times less efficient at cleaving type I or III
collagen (17). Similarly, MMP-14 is 5-7 times less efficient at
hydrolyzing type I collagen than MMP-1, whereas its gelatinolytic
activity is 8 times higher than that of MMP-1 (51).
To further investigate the role of the 189 exosite for macromolecular
substrate recognition, we docked a collagen triple helix to a
full-length collagenase (MMP-1). In addition to optimizing overall
contact areas of the substrate-enzyme complex, we were guided by the
following localized interactions: (a) the contact of the
collagen helix with the primary substrate recognition sites, including
the catalytic Zn2+; (b) the contact of the
collagen helix with the 189 exosite; and (c) the interaction
of the collagen hydroxyproline with His207. We used
published data for modeling the structures of the isolated components
(11, 55, 56). The most reliable and powerful conclusion from these
modeling studies is the orientation of the extended collagen peptide
relative to the enzyme. Earlier models postulated that the triple helix
makes major contacts with the first "blade" of the propeller-like
hemopexin domain (48). We conclude, however, that the triple helix will
not lie in the MMP active site oriented along the shortest route to the
hemopexin-like domain, which would bring it into contact with its first
blade. Instead, we propose that the substrate runs through the 188 exosite, leading to major contacts to blade 2 of the C-terminal
collagenase domain, consistent with the chimera mutant studies by
Nagase and co-workers (49). The extended contact of the collagen
substrate with the catalytic domain is consistent with a collagenolytic activity of the catalytic domain alone, as described for MMP-1 (57). On
the other hand, the conservation of the substrate exosite within MMP-1
and MMP-8 would suggest that the catalytic domain of MMP-8 should also
exhibit a collagenolytic activity that however has not been observed
(48). A second important consequence of these modeling studies is that
at least one of the collagen strands must be bent or arched by
~20o; an perfectly straight, rod-like collagen model
binding to the active site of MMP-8/1 remains ~6.5 Å distant from
the collagen exosite (Tyr189-Asn190). This
bending may be part of the unwinding mechanism of the collagen triple
helix necessary for its proteolysis (48). The occurrence of a
presumably functional cis-peptide bond at the type I collagen exosite
of collagenase 1 and 2 poses the question of its precise mechanism in
collagenolysis. The function of the 188 loop should be structurally
linked to either (a) the amino acid 188 side chain or
(b) the backbone conformation due to the cis-peptide bond.
Since the residue at the "nonglycine" position 188 of MMP-1 and
MMP-8 (Fig. 6) shows conserved similarity but not identity, a
contribution of option b seems likely. A detailed understanding of the 188-exosite's mechanism in collagen processing, and in particular the role of the amino at 188, awaits further experiments.
group of Ser209 and N
1 of
His207 of a symmetry related molecule; this interaction
suggests a model for recognition of hydroxyprolines present in
physiological substrates. We also identify a collagenase-characteristic
cis-peptide bond, Asn188-Tyr189, on a loop
essential for collagenolytic activity. The sequence conservation
pattern at this position marks this cis-peptide bond as a determinant
for triple-helical collagen recognition and processing.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Tyr189) could be
unambiguously identified. The conservation patterns of the sequence at
the cis-peptide bond position support the hypothesis that this
cis-peptide plays a critical role in substrate recognition mechanisms
specific to the collagenases I and II (MMP-1 and MMP-8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the active
site-directed inhibitors. RO200-1770,
2-hydroxy-5-phenyl-5-N-(4-(2-hydroxyethyl)-piperidyl)-4,6-pyrimidinedione;
RO204-1924,
2-hydroxy-5-phenyl-5-N-(4-(2-hydroxyethyl)-piperazyl)-4,6-pyrimidinedione;
I-COL043,
2-hydroxy-5-n-octyl-5-N-(4-(2-hydroxyethyl)-piperazyl)-4,6-pyrimidinedione;
RO206-0027,
2-hydroxy-5-phenyl-5-N-(4-paranitrophenylpiperazyl)-4,6-pyrimidinedione;
RO206-0032,
2-hydroxy-5-n-octyl-5-N-(4-paranitrophenylpiperazyl)-4,6-pyrimidinedione
pN1
bond have staggered orientations. This results in an arrangement where
all three rings are mutually perpendicular, as follows: the angle
between (the normal vectors) of the barbiturate and phenyl rings is
91°, between the barbiturate and the piperidyl rings is 103°, and
between the phenyl and piperidyl rings is 111°.
N3 coordination has
a favorable distance of 2.09 Å and highly symmetric
Zn2+-N3-C2 and
Zn2+-N3-C4 angles of 119° and
117°, respectively. Positioned where the catalytic water is expected
for peptidic substrates, a partial negative charge at the hydroxyl
O2 is stabilized by the
adjacent Glu198, thereby strengthening its binding to
Zn2+ (Figs. 2 and 3, Table
II). The enol form of the barbiturate is thus favored by the protein
matrix over the tautomeric keto form, which dominates in solution (37).
In addition, the polar
H1-N1-C6=O6 atoms of
the barbiturate (Fig. 3B) mimic the P1'-S1' antiparallel main chain interactions of a substrate (Fig. 3A). The amide
N1-H1 thereby is hydrogen bonded to the
carbonyl of Ala161, and the ketone
C6=O6 is stabilized by the amides of
Leu160 and Ala161. This latter interaction is
reminiscent of the oxyanion hole binding of serine proteinases,
although here there is no evidence of oxygen anion stabilization. The
C4=O4 ketone seems unlikely to contribute to
the binding energy for two reasons. First, unfavorable geometry (Table
I) precludes a role as a third ligand in
the Zn2+ chelation. More importantly, the
C4=O4 ketone would collide with the carbonyl
oxygen position of Pro217 at the "southern" rim of the
active site as defined by other MMP-8 structures (2, 14, 26). Instead,
the inhibitor induces a reorientation at the Pro217
position at an energy cost we discuss below.
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Fig. 2.
The RO200-1770 inhibitor bound to the active
site of MMP-8 (yellow). The
2Fo Fc electron
density map is contoured 1.0
over the mean. Pro217
(red) from a reference structure was superimposed to the
protein model (yellow) to illustrate the conflict of its
carbonyl oxygen with barbiturate binding. The figure was prepared by
using the program MAIN (34).
View larger version (16K):
[in a new window]
Fig. 3.
A, schematic representation of the
active site determinants for substrate recognition. B,
schematic representation of the interaction of the
2-hydroxypyrimidinedione with the MMP-8 active site. C,
schematic representation of the Zn2+-interaction geometry
of a hydroxamic acid-based inhibitor (batimastat) (2).
Zn2+ coordination geometry: bonds, angles, and dihedral angles
2(His197), and
N
2(His207) approximately in
plane with the Zn2+ ion, with
N
2(His201) and N3
lying above and below the basal plane, respectively (Table I).
Alternatively, the coordination can be described as a distorted square
pyramid where O2, N3,
N
2(His201), and
N
2(His207) form the basal
ligands (dihedral deviation from planarity 15°, Table I). The metal
ion lies outside of the basal plane but within 0.5 Å, and the fifth
ligand N
2(His197) forms the apex
of the pyramid Table I. (If considering O4 to be a sixth
ligand, the geometry may be described as a pentagonal pyramid with
O4 as basal ligand in addition to O2,
N3, N
2(His201), and
N
2(His207)).
2 and
N
1 of Arg222 are fixed by
hydrogen bonds to the carbonyl oxygen of Ala213 (3.3 Å)
and the O
of Tyr227 (3.3 Å), respectively.
Since most MMPs lack an equivalently stabilized Arg, MMP-8 has a
comparatively restricted S1' site.
View larger version (28K):
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Fig. 4.
Details of the histidine-phenyl stacking
interaction.
2 and C
values
measured as follows: His197 (0.4 Å, 0.3 Å),
His201 (0.3 Å, 0.3 Å), and His207 (0.6 Å,
0.7 Å). Consistent with this overall shift, the side chain of the
catalytic Glu198 is translated by 0.2 Å. This displacement
of the catalytic Zn2+ and its protein ligands is evidently
induced by inhibitor binding, as the net effect of the optimization of
barbiturate-Zn chelation geometry and the inhibitor orienting forces
arising from the other inhibitor-protein interactions.
1 and
C
2 positions.
Profiling of MMP inhibitors (IC50 [nM])
1 of the zinc ligand His207,
reminiscent of the charge relay system of serine-proteases. This
interaction thus bridges Pro217 and His207 from
one MMP-8 molecule with Ser209 of the neighboring enzyme.
To create this hydrogen bond, the Ser209 side chain adopts
a different
1 rotamer different from earlier MMP-8 structures. A
crystallographic 2-fold axis is located adjacent to Tyr216
and Pro217. The side chain of Asn218 is
reoriented compared with typical MMP-8 structures (where an intermolecular hydrogen to a symmetry related Thr129
exists) and forms a hydrogen bond with a symmetry-related
Tyr216 O
. None of the crystal contacts
interfere with expected peptidic binding sites. A symmetry-related
Gln133, however, forms a hydrophobic contact at a
depression bounded by Ile159 and Ser151, which
could serve as an alternative S2/S3 binding site. There is no direct
contact of the inhibitor with a symmetry-related protein molecule.
-helix
L191-H197EXXH201
L203.
The only restraint apparent for this structural framework is a
stabilizing hydrogen bond between carbonyl oxygen of Thr181
with the amide of Tyr189. Sequence comparison of this
segment with related MMPs reveals a subdivision within the MMP family.
Only collagenases 1 and 2 (MMP-1 and MMP-8) lack a glycine at position
188, a feature otherwise absolutely conserved, including nonhuman
species as well. We therefore predict that the
Glu188-Tyr189 peptide bond of MMP-1 also
adopts a cis-conformation. As exemplified by the crystal structure of
stromelysin 1 (MMP-3) (23, 43), Gly188 exhibits dihedral
angles (
,
) = (150°, 165°), which correspond to a
conformation allowed only for glycine. Therefore, glycine is conserved
at position 188 presumably to stabilize the local fold; conservation of
a nonglycine residue (MMP-1, MMP-8) suggests a function related to the
cis-peptide bond.
View larger version (73K):
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Fig. 5.
Cis-peptide bond between Asn188
and Tyr189. The cis-peptide bond is highlighted in
red. The figure was prepared by using the program MAIN
(34).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
N
2 atoms to the backbone carbonyl groups of
Leu222 and Thr215 (Asn218 and
Pro211 in MMP-8). The MMP-3 active site is thus
incompatible with binding the barbiturate ring, as reflected by the
overall lower binding constants. His224 is unique to MMP-3.
The observed progressive increase of binding affinity with enlarging
P1' or P2' residues is likely due to generally increasing hydrophobic
surface areas.
Asn218 will provide much of the
flexibility necessary for collagen substrate recognition (48), along
with the additional plasticity seen at the catalytic Zn2+
and its ligating residues. The generally weaker inhibition constants indicate that stromelysin 1 (MMP-3) does not possess the necessary plasticity in this segment for barbiturate binding.
His207 might provide an unexpected
opportunity for synthetic drug design. In particular, this interaction
offers the welcome possibility to deviate from peptide-like binding
patterns at a highly ordered position in the active site. Intriguingly,
since hydroxyprolines and other hydroxylated amino acids are present in
the physiological substrates of the extracellular matrix, we speculate
that this crystal contact may mimic a hydroxylated substrate
interaction. An alternative possibility that also would exploit the
common His-Ser motif would be an interaction with His201
N
1.
View larger version (29K):
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Fig. 6.
Local sequence comparison of related MMPs
around the collagen recognition exosite. All sequences are human
unless otherwise noted. The complete list of known MMP sequences shows
the same pattern that only MMP-1 or MMP-8 have nonglycine residues at
position 188. Sequences were derived from Swissprot.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Angelika Esswein, Valeria Livi, Ernesto Menta, and Gerd Zimmermann for synthesizing the compounds RO204-1924, I-COL-043, RO206-0027, and RO206-0032. We thank Dr. Christopher M. Overall for communicating site-directed mutagenesis results prior to publication.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We dedicate this work to Prof. H. Tschesche on the occasion of his 65th birthday.
§ To whom correspondence should be addressed. Tel.: 49-89-8578-2828; Fax: 49-89-8578-3516; E-mail: hbs@biochem.mpg.de.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M007475200
2 C. M. Overall, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MMP, matrix
metalloproteinase;
TACE, tumor necrosis factor converting
enzyme.
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