From the Center for Gene Therapy, Allegheny University of the
Health Sciences, Philadelphia, Pennsylvania 19102
A series of experiments were carried out to test
the hypothesis that the self-assembly of collagen I monomers into
fibrils depends on the interactions of specific binding sites in
different regions of the monomer. Six synthetic peptides were prepared
with sequences found either in the collagen triple helix or in the N-
or C-telopeptides of collagen I. The four peptides with sequences found
in the telopeptides were found to inhibit self-assembly of collagen I
in a purified in vitro system. At concentrations of 2.5 mM, peptides with sequences in the C-telopeptides of the
1(I) and
2(I) chain inhibited assembly at about 95%. The
addition of the peptide with the
2-telopeptide sequence was
effective in inhibiting assembly if added during the lag phase and
early propagation phase but not later in the assembly process.
Experiments with biotinylated peptides indicated that both the N- and
C-telopeptides bound to a region between amino acid 776 and 822 of the
(I) chain. A fragment of nine amino acids with sequences in the
2-telopeptide was effective in inhibiting fibril assembly. Mutating
two aspartates in the 9-mer peptide to serine had no effect on
inhibition of fibril assembly, but mutating two tyrosine residues and
one phenylalanine residue abolished the inhibitory action. Molecular
modeling of the binding sites demonstrated favorable hydrophobic and
electrostatic interactions between the
2telopeptide and residues
781-794 of the
(I) chain.
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INTRODUCTION |
Fibrillar collagens form the largest protein structures found in
complex organisms (see Refs. 1 and 2). The most abundant collagen
fibrils consist almost entirely of a single monomer of type I collagen.
The structure of the monomer was established several decades ago, but
the precise pattern of packing of the monomer into fibrils has not
been defined and remains controversial (3-17).
Type I collagen is similar to other fibrillar collagen in that it
is first synthesized as a soluble procollagen containing N-propeptides
and C-propeptides (see Ref. 2). The propeptides are cleaved by specific
N- and C-proteinases to generate the monomers that comprise collagen
fibrils. The two
1(I) and one
2(I) chains of a monomer of type I
collagen are primarily comprised of about 338 repeating tripeptide
sequences of Gly-Xaa-Ybb in which Xaa is frequently proline and Ybb is
frequently hydroxyproline. The ends of the
1(I) and one
2(I)
chains consist of short telopeptides of about 11-26 amino acids per
chain. The distribution of hydrophobic and charged resides in the Xaa
and Ybb positions in the triple-helical domain define 4.4 repeats or
4.4 D periods of about 234 amino acids each. In longitudinal sections,
the monomers are arranged in fibrils in a head-to-head-to-tail
orientation with a gap of about 0.6 D periods and, therefore, repeat of
5 D periods. The continuity of the fibrils is maintained by many of the
monomers being staggered by 1, 2, 3, or 4 D periods relative to the
nearest neighbor so as to generate gap and overlap regions. However,
there are conflicting data from electron microscopy and x-ray analysis about the lateral packing of the monomers. One view is that the monomers are laterally packed in a tilted quasi-hexagonal lattice (4,
14). A related view is that the fibrils consist of "compressed" microfibrils that are comprised of monomers coiled into a rope-like pentameric structure (3, 6). Still another view is that the lateral
packing of the collagen in many fibrils is either liquid-like or a
biological equivalent of a liquid crystal (12, 13).
One experimental approach to defining the lateral packing of the
monomers was to observe the initial assembly of monomers into fibrils.
Early experiments (1, 11, 15) on the reassembly of fibrils from
collagen extracted from tissues with acidic buffers suggested that the
first structures formed were linear strands of monomers bound by 0.4 D
period overlaps (4 D staggers). Other observations with extracted
collagens suggested the initial stages involved assembly of structures
similar to pentameric microfibrils (1, 15, 16). Subsequently, a system
was developed for studying assembly of type I collagen fibrils de
novo by enzymic cleavage of a purified soluble precursor of
procollagen under physiological conditions (18-21). Because thick
fibrils were generated in the system at 30-32 °C, it was possible
to use dark-field light microscopy to follow the growth of the fibrils
through intermediate stages (21). The first fibrils detected had a
blunt end and a pointed end or tip. Initial growth of the fibrils was
exclusively from the pointed or
tip. Later,
tips appeared on
the blunt ends of the fibrils, and the fibrils grew from both
directions. Scanning transmission electron microscopy indicated that
both the
tips and
tips were near-paraboloidal in shape (22).
Also, the monomers were oriented with their N termini directed toward
the tips. Subsequent experiments in the same system with type II
collagen suggested that the fibrils also grew from pointed tips.
However, the monomers were oriented with a C termini directed toward
the tips (23). Also, the fibrils contained central regions in which the
monomers were packed symmetrically. Recent observations indicate there may be differences between collagen fibrils assembled in
vitro and those assembled in vivo (24). In particular,
collagen I fibrils assembled in vitro are exclusively
bipolar, but fibrils from tissues are both bipolar and unipolar. Also,
intermediates such as pNcollagen may participate in the initial steps
of fibril assembly. All the data, however, are consistent with the
conclusion that the fibrils grow from pointed tips.
Three different models were proposed to explain the growth of fibrils
from near-paraboloidal tips. One model (25) was based on the assumption
that the initial core of the fibril was a pentameric microfibril and
that the fibril grew by the addition of monomers in a helical pattern.
Simulations of the model suggested that as little as two specific
binding steps were required, first for assembly of the microfibrillar
core and then a structural nucleus with about the same diameter as the
final fibril. After assembly of the structural nucleus, the fibril grew
from the paraboloidal tip by the addition of monomers through only one
of the two binding steps. A second and related model (26) suggested
that assembly began with formation of an undefined inner core, and then
monomers were added in spiral strands to generate the paraboloidal
tips. The second model had the advantage that it more readily than the first model accounted for x-ray diffraction data that indicated that
some fraction of monomers in fibrils were laterally packed in a tilted
quasihexagonal lattice (4, 14). In contrast to the first two models, a
third model (27) was developed in which monomers were assembled by a
process involving only aggregated limited diffusion. The third model,
therefore, assumed that the assembly of monomers into fibrils was
similar to processes such as electrochemical depositions or perhaps
formation of snowflakes and that the process did not require the
presence of specific binding sites on the monomers.
Here, we have developed a series of experiments on the assumption that
the assembly of collagen I monomers into fibrils depends on the
interactions of specific binding sites in different regions of the
monomers. To test this assumption, we examined the effects of synthetic
peptides on the assembly of fibrils de novo.
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MATERIALS AND METHODS |
Design and Synthesis of Peptides--
The synthetic peptides
were synthesized and purified by a commercial concern (American Peptide
Company, Inc.). Homogeneity and stability of the peptides in
experimental conditions were assayed by reverse-phase
HPLC.1 All the peptides were
freely soluble in the fibril formation buffer. The pH of peptide
solutions was monitored with a solid state micro-pH electrode
(Beckman).
Assay of Fibril Assembly--
Assembly of collagen I into
fibrils de novo was assayed under conditions employed
previously (18-21, 23). In brief, 14C-labeled type I
procollagen was recovered from the medium of cultured human skin
fibroblasts and was purified with two chromatographic steps to
homogeneity. The type I procollagen was processed to pCcollagen I by
cleavage with procollagen N-proteinase purified from organ cultures of
chick embryo tendons. The pCcollagen was then isolated on a gel
filtration column. Fibril assembly was assayed in a 20-µl reaction
volume in a 250-µl plastic centrifuge tube sealed with a plunger and
containing a physiological bicarbonate buffer, 30 µg/ml pCcollagen I,
and 15 units/ml procollagen C-proteinase purified from chick embryo
tendons. Potentially inhibitory peptides were added to the reaction
mixture to final concentrations of 0.5-2.5 mM in 5 µl of
buffer. After incubation for 0.5 to 24 h at 37 °C, the sample
was centrifuged 13,000 × g for 10 min. The pellet and
supernatant fractions were separated by electrophoresis on a 7.5%
polyacrylamide gel in SDS, and the gel was assayed either with a
phosphor storage imager (STORM, Molecular Dynamics) or by staining with
colloidal Coomassie Blue (Brilliant Blue; Sigma) and analysis with a
densitometer (Personal Densitometer, Molecular Dynamics).
Alternatively, the reaction was carried out in a sealed chamber on a
microscopic slide and followed by dark-field light microscopy (Zeiss
model 009).
Localization of Binding Sites on Collagen--
Type I collagen
extracted with 0.5 M acetic acid from mouse skin was
digested with pepsin, and the
chains were separated by gel
electrophoresis in SDS. To generate CNBr peptides, gel slices
containing
1(I) and
2(I) chains were placed into tubes, and
chains were digested with 10 mg/ml or 200 mg/ml CNBr in 70% formic
acid at room temperature overnight. The gel slices were equilibrated
with 0.05 mM Tris-HCl buffer (pH 6.8) over 3 h. Gel
pieces containing CNBr peptides derived from individual collagen
chains were transferred into the wells of the second gel prepared
with a 6% polyacrylamide stacking gel and 12% polyacrylamide separating gel with 0.5 M urea. After electrophoresis, the
gel was electroblotted overnight at 4 °C onto a nitrocellulose
filter (Millipore). To study binding of telopeptides to collagen
fragments, the method described by Fujisawa et al. (28) was
used. The filters were blocked with 1% bovine serum albumin (Sigma)
and then incubated overnight with 20 ml of 5 µg/ml peptide that was
substituted at the N terminus with biotin (synthesized for us by
American Peptide Company, Inc.). The filter was washed three times with
Tris-buffered saline containing 0.02% Tween and incubated 30 min with
a 1:30,000 dilution of horseradish peroxidase conjugated with avidin
(Sigma). The bands were detected by chemiluminescence (ECL; Amersham
Pharmacia Biotech) after exposure to an x-ray film for 3 to 10 min.
To generate vertebrate collagenase fragments, 3 µg of type I
pCcollagen from cultured human fibroblasts was cleaved with 10 µg/ml
of vertebrate collagenase from cultured rat skin fibroblasts (generous
gift from John J. Jeffrey, Department of Medicine, Albany Medical
College) for 3 h at 25 °C in a volume of 40 µl of 50 mM Tris-HCl, 10 mM CaCl2 and 100 mM NaCl, pH 7.4. To remove the C-propeptide, 2 µl of a
mixture of trypsin (1 mg/ml) and chymotrypsin (2.5 mg/ml) in the same
buffer was added, and the sample was incubated at 20 °C for 2 min.
The reaction was stopped with 0.5 mg/ml soybean trypsin inhibitor
(Sigma). The sample was then separated by electrophoresis on a 10%
polyacrylamide gel in SDS and processed with the same protocol as the
CNBr fragments.
Competition Assay--
To define the telopeptide binding site
with a competitive assay, 96-well titration plates (Immulon 3, Dynatech
Laboratories, Inc.) were used. Seventy microliters of the solution
containing 3 µg of pepsin-treated monomeric collagen (Vitrogen 100, Collagen Biomaterials) in 0.01 N HCl was added to each
well, and the plates were dried at room temperature. As a control,
wells covered with bovine serum albumin were used. The plates were
rinsed with sterile phosphate-buffered saline and nonspecific binding
sites were blocked with 1% bovine serum albumin. For the competition
assay, pre-mixed samples were prepared that contained 50 µM biotinylated F2 or F3 peptide with 10-500
µM peptide
1-776/796. As a negative control, peptide
2-218/233 (F1) was used at concentrations from 10 to 500 µM. The peptide mixtures were added into wells containing immobilized collagen, and the samples were incubated at 25 °C for
12 h. The plates were washed with PBS containing 0.05% Tween. To
detect biotinylated peptides bound to collagen surface, streptavidin conjugated to alkaline phosphatase (Bio-Rad) was added at 1:20,000 dilution. After 2 h of incubation, the plates were washed with PBS
containing 0.05% Tween, and the developing reagent 3 mM
p-nitrophenyl phosphate in 0.05 M
Na2CO3, 0.05 mM MgCl2
(pH 9.5) was added. After 2 h of incubation, the reaction was
measured with microtiter plate reader (Dynatech Laboratories) using a
405-nm filter.
Computer Modeling of Binding Sites--
Molecular modeling was
performed on a Silicon Graphic (Indigo 2) computer system using the
SYBYL software package, Version 6.3 (Tripos, Inc.) The model of the
collagen I triple helix fragment including sequence from
1-766/801
and
2-766/801 was carried out as described by Chen et al.
(29). The model of F7 peptide was created using SYBYL/Biopolymer
module. All the models were energy-minimized using a conjugate gradient
method and subjected to repeating cycles of molecular dynamics using
Kollman force field and united atoms (30). To analyze interaction of F7
peptide with the collagen I binding region, intermolecular energy of
interaction was analyzed to identify possible binding conformations.
Surface calculations, lipophilicity potential, and electrostatic
potential of the molecules were analyzed using SYBYL/Molcad module.
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RESULTS |
Assays of Inhibition of Fibril Formation with Synthetic
Peptides--
Six synthetic peptides (Fig.
1) were prepared on the basis of two
general considerations. (a) Extensive previous work (see Ref. 1) had demonstrated that the telopeptides were required to be
present on the monomers to generate tightly packed fibrils, and
(b) the self-assembly of collagen monomers is entropy-driven (see Ref. 19), and therefore, any specific binding sites in the triple
helix are likely to be found in hydrophobic sequences. As indicated in
Fig. 1, one peptide (F1) contained relatively hydrophobic sequences
found near the end of the D1 period of the
2(I) chain, a second
peptide (F5) had hydrophobic sequences found near the middle of the D4
period of the
2(I) chain. F6 contained sequences that spanned the
vertebrate collagenase cleavage site of Gly-Ile in the D4 period of the
1(I) chain. The remaining three peptides had the sequences of the
1N-telopeptide,
1C-telopeptide, and
2C-telopeptide. Assays by
reverse-phase HPLC demonstrated that none of the peptides were degraded
under experimental conditions used here (data not shown). The peptides
were added in concentrations of 2.5 mM to the system for
assaying fibril formation by the cleavage of pCcollagen with
C-proteinase (18-21, 23). As indicated in Fig.
2 and Table
I, the peptides F1 and F6 caused little
if any inhibition of fibril assembly. Peptides F2, F3, and F4 almost completely inhibited fibril assembly, whereas F5 inhibited assembly by
about 50% under the conditions of the experiment. Inhibition of fibril
assembly with the peptide F3 was directly demonstrated by following the
reaction in a sealed chamber with dark-field light microscopy. No
fibrils appeared when F3 was present in a concentration of 2.5 mM (Fig. 3, right
panel). The specificity of the effects was demonstrated by
preparing a peptide in which the same amino acids found in F3 were
assembled in a random sequence. As indicated in Fig. 2 (bottom
panel), the peptide with the scrambled sequence (SC/F3)
had no effect on fibril assembly. Assays in which the concentration of
the peptide F3 were varied indicated that fibril assembly was inhibited
about 45% with a concentration of about 1.5 mM and almost
100% with 2.5 mM (Table I).

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Fig. 1.
Schematic of the structure of a type
I-collagen molecule divided into D periods, N-telopeptides, and
C-telopeptides. Sequences above and below the model indicate
the sequence of the peptides tested here as inhibitors of fibril
assembly. Symbols: ( ), no effect on fibril assembly; (+), about 50%
inhibition of fibril assembly in the concentration of 2.5 mM; and (++), complete inhibition of fibril assembly in the
concentration of 2.5 mM under the conditions used here;
NT, N-terminal telopeptides; CT,
C-telopeptides.
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Fig. 2.
Fibril formation in the presence of synthetic
peptides. 14C-Labeled pCcollagen I (30 µg/ml) was
incubated with C-proteinase (15 units/ml) for 24 h at 37 °C.
F1, F2, F3, F4, F5, and F6 peptides were added to the reaction mixture
at concentrations of 2.5 mM. Fibrils were separated from
collagen monomers by centrifugation and analyzed by gel electrophoresis
in SDS. Panels, images from a phosphor storage plate of
three electrophoretic gels. Symbols: P, pellet fraction;
S, supernatant fraction; C, control sample;
SC/F3, same peptide as F3 with scrambled sequence
(YGAGFDDGDYGYRYG).
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Fig. 3.
Darkfield analysis of collagen I fibrils.
pCcollagen was incubated with C-proteinase at 37 °C for 24 h in
a sealed chamber in the presence (+) or absence ( ) of 2.5 mM F3 peptide. Fibrils were photographed using a
microscope with dark-field attachment. Magnification, 300×.
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The
2C-telopeptide (F3) Inhibits Fibril Assembly if Added to the
Lag Phase and Early Propagation Phase--
To define the kinetics for
inhibition of fibril assembly by the telopeptides, the peptide F3 was
added in a concentration of 2.5 mM during the lag phase,
early propagation phase, mid-propagation phase, or late propagation
phase of fibril assembly (Fig. 4). The
peptide inhibited fibril formation if added during the lag period. It
partially inhibited if added during the early propagation phase, and it
had little of any effect if added during the mid-propagation phase of
fibril assembly.

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Fig. 4.
Effects of adding the peptide F3 at varying
time points during fibril assembly. Assays as in Fig. 2. Symbols:
, fibril assembly under control conditions; arrows, times
when 2.5 mM F3 was added to parallel samples and then the
incubation continued for 24 h; and , values observed at 24 h..
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Location of the Telopeptide Binding Site in the Triple Helix of
Collagen I--
To test the specificity of the binding of the peptides
to collagen, a series of hybridizations were carried out with filter blots of proteins and a biotinylated derivative of the peptide F3. As
indicated in Fig. 5, the biotinylated
peptide F3 did not bind to any component of bovine serum. However, it
bound to
1(I) chains,
chains that included
1(I) chains,
pC
1(I) chains, and pC
1(II) chains. The same peptide bound to CNBr
peptides (Fig. 6). As indicated in Fig.
6B, the biotinylated derivatives bound to the
1(I) chain
but not the
2(I) chain. As also indicated in Fig. 6B, the
biotinylated telopeptides bound to CB7 of the
1(I) chain that
contains amino acid residues 552-822. There was no apparent binding to
any of the other cyanogen bromide fragments.

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Fig. 5.
Binding of the biotinylated peptide F3 to
filter blots of proteins. The proteins were separated by
polyacrylamide gel electrophoresis in SDS and electroblotted onto
filters. The filters were then hybridized with the biotinylated
2C-telopeptide followed by assay of the washed filter with
streptavidin alkaline phosphatase. Left panel, filter
stained with Coomassie Blue. Right panel, filters hybridized
with biotinylated F9. Lane 1, pepsinated type I collagen
from bovine skin. Lane 2, bovine serum proteins.
Lane 3, pCcollagen II. Lane 4, pCcollagen
I.
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Fig. 6.
Binding of biotinylated telopeptides to the
(I) chain and the CB7 fragment chain. Conditions as in Fig. 5.
Panel A, Coomassie Blue-stained filter of 1(I) and
2(I) chains and CNBr peptides. Panel B, filters probed
with biotinylated telopeptides and streptavidin conjugated to
peroxidase. Symbols: CNBr +, 1(I) or 2(I) chains digested with
cyanogen bromide; F2, F3, F4, and NA2, separate filters probed with
biotinylated C-telopeptide of the 1(I) chain, C-telopeptide of
2(I) chain, N-telopeptide of 1(I) chain, and N-telopeptide of
2(I) chain.
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To further define the binding site in the triple helix, vertebrate
collagenase A and B fragments of type I collagen were prepared and
hybridized with the biotinylated telopeptides. As indicated in Fig.
7 (middle and right
panels), the peptides bound specifically to the B fragment of the
1(I) chain. Since vertebrate collagenase cleaves the two
chains
of type I collagen between residues 775 and 776 (1), the binding of the
peptides to both CB7 and the B fragments of the
1(I) chain indicated
that the binding site is between amino acid 776 and 822 of the
1(I)
chain.

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Fig. 7.
Binding of biotinylated telopeptides to
collagenase A and B fragments of type I collagen. Left
panel: Coomassie-stained polyacrylamide gel of 1(I) and 2(I)
chains and vertebrate collagenase fragments. Middle and
right panels, filters probed with biotinylated telopeptide
and streptavidin conjugated to horseradish peroxidase. Symbols:
VC /+, samples with or without prior digestion with
vertebrate collagenase; T, CH, bands of trypsin
and chymotrypsin used to convert the procollagen into collagen. As
indicated, there is some reactivity of the biotinylated telopeptides
with large amounts of trypsin and chymotrypsin. Other symbols as in
Fig. 6.
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Defining the Critical Residues in the
2C-telopeptide
(F3)--
Further experiments were primarily concentrated on the F3
peptide with the sequence of the C-telopeptide, because it was
relatively short and somewhat repetitive in sequence (Fig. 1).
To define the critical sequences within the peptide F3, several
derivatives were prepared. Two peptides that were 9-mers overlapping the central region of the sequence (F7 and F8) were equally effective as the intact F3 (Figs. 1 and 8 and Table
I). Smaller effects on inhibition of fibril assembly were seen with two
other fragments (F9 and F10) (Figs. 1 and 7 and Table I). Of special
interest was that mutating the two aspartates in a 9-mer peptide (F7)
to serine residues (F7/A) had no effect on inhibition of fibril
assembly. However, mutating two tyrosines and one phenylalanine in the
same sequence to serine residues (F7/B) abolished inhibitory effects (Figs. 1 and 8 and Table I).

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Fig. 8.
Assay of inhibition of fibril formation with
peptides that are fragments and modified version of the -telopeptide
(F3). See Fig. 1 and Table I for summary. Symbols: P,
pellet fraction; S, supernatant fraction; C,
control sample.
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Defining the Critical Sequences in the
1(I) Chain--
To map
the binding site in the triple helix still further, a peptide was
prepared with the sequence of the amino acids in positions 776 to 796 of the
1(I). The sequence 776 to 796 was selected primarily because
it was the most hydrophobic sequence in the region between 776 and 822 that was defined by the experiments with the CNBr peptides and the
collagenase fragments (Fig. 9). The
peptide
1-776/796 was then used in a competition binding assay in
which collagen was bound to the wells of a microtiter plate, and the
plates were incubated with a fixed concentration of one of the
biotinylated telopeptides and varying concentrations the peptide
1-776/796. The binding of the biotinylated peptide was then assayed
by incubation with streptavidin-alkaline phosphatase. As indicated in
Fig. 10, the peptide
1-776/796 effectively competed with binding the C-telopeptides of
the
1(I) chain (peptide F2) and the
2(I) chain (peptide F3).
There was no competition for the binding site between biotinylated
telopeptides and F1. Similar results (not shown) were obtained with the
binding of the two N-telopeptides.

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Fig. 9.
Schematic of the binding site in the triple
helical domain of the (I) chain as defined by the experiments with
CNBr and vertebrate collagenase fragments (Figs. 6 and 7).
Bottom of figure indicates that the binding of the
C-telopeptides to the region is consistent with one quarter D stagger
of monomers and fibrils. The underline sequence of 1-776
to 796 was used as competitive peptide to further define the binding
site (see Fig. 10).
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Fig. 10.
Competition by the peptide -776/796 for
the binding of the C-telopeptides to type I collagen. Type I
collagen was bound to microtiter plates and then incubated with a
mixture of a biotinylated telopeptide and varying concentrations of the
competitor peptide 1-776/796. The washed filters were then assayed
by reaction with streptavidin-alkaline phosphatase. Left
panel, competition for binding of the biotinylated C-propeptide of
the 1(I) chain (peptide F2). Right panel, competition for
the binding of the C-telopeptide from the 2(I) chain (peptide
F3).
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Molecular Modeling of the Two Binding Sites--
To model the
binding of one of the telopeptides to the triple helix, the SYBYL
program was first used to model the conformation of one of the
nine-amino acid fragment (F7) of the
2C-telopeptide that inhibited
fibril assembly (Figs. 1 and 8 and Table I). Because the aromatic Tyr
and Phe residues tended to form a hydrophobic stack on one side of the
polypeptide chain, a single conformation was favored (Fig.
11, A and B).

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Fig. 11.
Computer model of the binding interaction
between a nine-amino acid peptide sequences from the C-telopeptide of
the 2(I) chain and the triple helix of type I collagen in the region
of amino acid -766 to 801. A, display of hydrophobic
residues. B, display of electrostatic charges.
Red indicates positive charges and, purple
indicates negative charges. 1-Arg 780, 1-Val 783, and 1-Arg
792 are shown to define the region of interaction.
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Binding of F7 to the region between residues 781 to 794 of
1(I)
chain was analyzed. The possible binding conformation was interactively
identified using the DOCK command. The refined model demonstrated a
favorable interaction of hydrophobic groups (Fig. 11A)
and electrostatic groups (Fig. 11B).
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DISCUSSION |
The results here resolve a critical question about the
self-assembly of type I collagen into fibrils. If the assembly does not
depend on specific interactions of binding sites in the monomers, as
recently suggested by Parkinson et al. (27), all the
peptides tested here should either have had no effect on fibril
assembly or inhibited the process to about the same degree. Instead,
the results demonstrated that several peptides specifically inhibited the process. Consistent with previous observations (see Ref. 1), peptides with sequences found in the telopeptides were the most effective. The telopeptide that was most extensively studied, the
C-telopeptide of the
2(I) chain, completely prevented fibril assembly if added at or before the first one-third of the lag phase but
had much less effect thereafter. Therefore, the binding of the
2C-telopeptide, probably in concert with
1C-telopeptide, is
critical for early steps in the assembly process such as formation of a
structural nucleus that is essential for further growth of the fibrils.
The binding of the C-telopeptides to the region that encompasses
residues 776 to 796 places the monomers in quarter D-period stagger
(Fig. 9). Therefore, the binding could initiate assembly of a
Smith-type pentameter microfibril (see Refs. 1 and 25). In contrast,
the binding of the N-telopeptides to about the same region of the
1(I) chain (defined here as residues 776 to 822) places the
D-periods out of register by one-third or more of a D-period of 234 residues. Therefore, the binding of the N-telopeptides cannot generate
the 0D, 1D, 2D, 3D, and 4D staggers that are found among many nearest
neighbors in fibrils assembled in vivo. Also, the binding of
the N-telopeptides does not accurately align the monomers for formation
of the major covalent cross-link that forms between the Lys residue at
position 9 of the N-telopeptide and the Lys residue at 930 of the
1(I) chain (1, 36). Accordingly, there are several possible
explanations for the observed binding of the N-telopeptides. One is
that the binding occurs only with linear N-telopeptides such as those
used here but not with N-telopeptides in the unusual hairpin
conformation that is present in the native molecule (31-35) and that
is essential both for assembly into well ordered fibrils and correct
cross-linking (36). A second explanation is that binding of the
N-telopeptides generates aberrant structural nuclei that cannot grow
into fibrils and that resembles the "overshoot" structures seen in
the assembly of tobacco mosaic virus (37). Preliminary assays with an
optical biosensor indicated that the dissociation constant for the
binding of the
1C-telopeptide is about 5 × 10
6
M, and the dissociation constants of the two N-telopeptides
are about an order of magnitude
greater.2 Therefore, aberrant
structures assembled by binding through the N-telopeptides may have a
short half-life and may rapidly dissociate into monomers that initiate
fibril assembly through binding of C-telopeptides. A third possibility
is that binding through the N-telopeptides does not play an important
role in fibril assembly until a core of a microfibril is formed, and it
is only important for lateral growth of the fibril. The last suggestion
is consistent with one of the proposed helical models for growth of
microfibrils from paraboloidal tips (25). The model required one
specific binding step governed by one rate constant
(k1) for assembly of monomers in a 1D-stagger to
form a Smith-type microfibrillar core and to regulate longitudinal
growth of the fibril. It required a second binding step governed by a
smaller rate constant (k2) to initiate growth of
a new layer of helical sheets of monomers on the microfibrillar core
and thereby to regulate lateral growth of the core.
The results here demonstrated that the binding of the C-telopeptide of
the
2(I) chain to residues 776 to 796 of the
1(I) chain was
directed primarily by hydrophobic interactions, since mutating two
tyrosine residues and one phenylalanine residue in a nine-amino acid
fragment abolished all effects on fibril formation, whereas mutating
two aspartate residues had no effect. The modeling experiments
indicated that there were conformations of the peptide and the triple
helix that allowed good hydrophobic and electrostatic interactions
between the nine-amino acid fragment
2C-telopeptide and the region
between 776 and 796. The region contains the C-terminal half of the
vertebrate collagenase cleavage site that previously has been
designated as a relatively flexible region of the collagen triple helix
(38). Also, Bhatnagar et al. (39) recently examined a
synthetic peptide with amino acid residues 776 to 780 from the region
and found that it had a high potential to form a stable
-bend with
the central GIAG sequence that begins in residues 775. Moreover, they
demonstrated (38) that the peptide inhibited the binding of fibroblasts
to collagen at a concentration of 7.2 × 10
6
M. Therefore, the sequence of amino acids in the region may
take part in a large number of different binding interactions.
Finally, it is apparent that given the specificity of the binding
interactions, the sites defined here provide interesting targets for
peptides, peptidomemetics, or related compounds that may inhibit the
assembly of collagen fibers in pathologic fibrotic conditions. The
ability to model binding sites in the triple helix and the conformation
of short fragments of the telopeptides provides a rational route for
developing inhibitors. The competitive assays on microtiter plates
provide a means of high throughput screening for large libraries of
potential inhibitors.