From the Institut de Biologie et Chimie des
Protéines, UMR 5086 CNRS-UCBL1, 69367 Lyon cedex 07, France,
the ¶ Departments of Pathology and Biomolecular Chemistry,
University of Wisconsin, Madison, Wisconsin 53706, the
European Molecular Biology Laboratory, Hamburg Outstation, 22603 Hamburg, Germany, the ** Institute of Crystallography,
Russian Academy of Sciences, 117333 Moscow, Russia, and the
Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 38027 Grenoble cedex 1, France
Received for publication, October 23, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Procollagen C-proteinase enhancer
(PCPE) is an extracellular matrix glycoprotein that can stimulate the
action of tolloid metalloproteinases, such as bone morphogenetic
protein-1, on a procollagen substrate, by up to 20-fold. The PCPE
molecule consists of two CUB domains followed by a C-terminal NTR
(netrin-like) domain. In order to obtain structural insights into the
function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All
three approaches showed PCPE to be a rod-like molecule, with a length
of ~150 Å. Homology modeling of both CUB domains and the NTR domain
was consistent with the low-resolution structure of PCPE deduced from
the small angle x-ray scattering data. Comparison with the
low-resolution structure of the procollagen C-terminal region
supports a recently proposed model (Ricard-Blum, S., Bernocco, S.,
Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869) for the
mechanism of action of PCPE.
Most extracellular matrix proteins are modular in structure (1,
2), including procollagen C-proteinase enhancer
(PCPE,1 molecular mass ~50
kDa), which consists of two CUB domains followed by a C-terminal
NTR domain (3, 4). One function of PCPE is to enhance the activities of
tolloid metalloproteinases (BMP-1, mTLD) (5-7), during procollagen
processing, by up to 20-fold (8-10). Tolloid metalloproteinases are
involved in a variety of morphogenetic events such as processing of
procollagens (11-15) (leading to fibril assembly) and prolysyl
oxidases (16, 17) (initiating covalent cross-linking in collagens and
elastin). They also cleave probiglycan (18), laminin 5 (19, 20)
(affecting keratinocyte adhesion/migration, Ref. 21) and chordin/SOG
(22, 23) (controlling dorso-ventral patterning). Enhancement of tolloid proteinase activity, on a procollagen I substrate, by PCPE has been
shown to be a property of the CUB domain region (3, 9, 10). Possible
roles of PCPE in tolloid proteinase processing of non-procollagen
substrates are unknown.
CUB domains are found exclusively in extracellular and
plasma membrane-associated proteins. In addition to PCPE and the
recently discovered PCPE2 (24), these include the tolloid
metalloproteinases BMP-1/mTLD, mTLL-1, and mTLL-2 (6, 7, 23), the
metalloproteinase ADAMTS13 (25), the complement serine proteinases C1r,
C1s, MASP1, MASP2, and MASP3 (26, 27) and the membrane serine
proteinases enteropeptidase/enterokinase (28) and matriptases-1 and -2 (29, 30). Further extracellular proteins include the spermadhesins (31), proteins consisting of single CUB domains involved in mammalian
fertilization, the inflammation-associated protein TSG-6 (32), the
growth factor PDGF-C/fallotein (33), and salivary agglutinin/gp-340/DMBT1 (34). Attractin, which exists in both secreted
and membrane-bound forms, displays dipeptidyl peptidase activity and is
involved in T cell clustering, skin pigmentation, and the control of
energy metabolism (35). Further membrane-associated proteins include
cubulin (36), a multi-CUB domain protein involved in intestinal
absorption of cobalamin (vitamin B12) as well as renal
protein re-absorption, and the neuropilins (37), involved in axonal
guidance and angiogenesis.
A number of roles for CUB domains have been assigned in protein-protein
and protein-carbohydrate interactions. In PCPE, the CUB domains bind to
the C-propeptide region of fibrillar procollagens (9), as well as
elsewhere in the procollagen molecule (38). In C1r and C1s, CUB domains
are required for C1s-C1r-C1r-C1s tetramer formation as well as
interaction of the tetramer with the collagen-like regions of C1q (27).
Similar roles have been demonstrated for the CUB domains of MASP-1 and
MASP-2 (as well as its alternatively spliced form Map19), in homodimer
formation and binding to the collagen-like region of mannan binding
lectin (39, 40). Some spermadhesins display carbohydrate binding
activity (31). In cubulin, CUB domains are involved in binding to
albumin and to the intrinsic factor-cobalamin complex (36), while in
neuropilin-1 CUB domains are required for binding to semaphorin 3A
(37).
The NTR module (4) occurs solely in extracellular proteins, whose
functions are sometimes similar or complementary to those of
CUB-containing proteins. The NTR family includes the netrins (41)
(proteins involved in axonal guidance), complement proteins C3, C4, and
C5 (26), secreted frizzled-related proteins (42) (mediators of Wnt
signaling), WFIKKN (43) (a protein containing multiple
protease-inhibitory modules) and TIMPs (44). Possible functions
for NTR domains are based on their homologies with the N-terminal
active domain of TIMPs. In the case of PCPE, it has recently been shown
that fragments corresponding to the free NTR domain are present in the
conditioned medium of human brain tumor cells, where they appear to
have moderate TIMP-like activity (45). While it has been speculated (4,
42, 43) that other NTR-containing proteins might also show TIMP-like
activity, direct experimental evidence for this is lacking.
Nevertheless, the possibility that PCPE contains domains that can
either enhance (CUB) or inhibit (NTR) different metalloproteinases is
intriguing. A further possibility is that the NTR and/or CUB domains in
PCPE are involved in the observed effects of this protein on the
control of cell growth (46-48), as has also been observed for TIMPs,
independent of metalloproteinase inhibitory activity (44).
So far the only CUB-containing proteins for which high resolution
three-dimensional structures have been determined are the spermadhesins
(31). While this has permitted the molecular modeling of individual CUB
domains in various proteins (32, 37, 49, 50), there exists at present
no information on how contiguous CUB domains might be arranged in three
dimensions. High-resolution structures of TIMPs (51, 52) permit
molecular modeling of the NTR domain. Here we use a variety of
biophysical techniques, together with molecular modeling, to determine
the low-resolution structure of PCPE. We show that the molecule is
highly elongated, and this gives insights into how PCPE enhances
procollagen C-proteinase activity.
Expression and Purification of Recombinant PCPE--
The 1474-bp
cDNA clone KT11 (3) was used as the template for PCR amplification
of sequences corresponding to full-length human PCPE, minus
signal peptide sequences, using forward primer 5'-ACTGTCAGCTAGCACAGACCCCCAACTACACCAGACCC-3' and reverse primer 5'-GCATGCGGCCGCAGTCCTGGGACGCAGCAG-3', which contained a
NheI or NotI site, respectively, to facilitate
subsequent cloning steps. The ~1.3-kb PCR product was ligated into
pGEM-T, sequenced fully on both strands to ensure an error-free clone,
excised by digestion with NheI and NotI and
ligated in-frame between the NheI and NotI sites
of the vector pCEP-Pu/BM40s (53). Excision of fused BM40-PCPE sequences
from the latter construct with KpnI and NotI and
insertion between the KpnI and NotI sites of
expression vector pcDNA3.1 (Invitrogen) yielded a construct
designed to produce a translation product differing from native PCPE
only in the replacement of the native signal peptide by the BM40 signal
sequence (for enhancement of secretion). Proper insertion of sequences
was verified by DNA sequencing of insert-vector junctions.
Human 293 embryonic kidney cells, purchased from the American Type
Culture Collection (Manassas, VA) and maintained in growth medium
consisting of Dulbecco's modified Eagle's medium (DMEM)/10% fetal
bovine serum (14), were transfected with the PCPE/pcDNA3.1 expression vector, using LipofectAMINE, according to the
manufacturer's protocols (Invitrogen). Two days post-transfection,
cells were placed in growth medium containing 500 µg/ml G418
(Invitrogen), selected for approximately 2 weeks, then G418-resistant
clonal lines were picked with cloning cylinders and maintained in
growth medium containing 250 µg/ml G418. Confluent monolayers of the clonal line determined by Western blotting to produce the highest levels of recombinant PCPE (rPCPE) were washed three times with phosphate-buffered saline and incubated in Dulbecco's modified Eagle's medium containing 250 µg/ml G418 and 40 µg/ml soybean trypsin inhibitor (Sigma) for 24 h. Medium was harvested and
centrifuged to remove cell debris, and protease inhibitors were added
to final concentrations of 10 mM EDTA, 1 mM
4-aminobenzamidine dihydrochloride, 1 mM
N-ethylmaleimide, and 0.5 mM
phenylmethylsulfonyl fluoride. Conditioned medium, which was typically
found to contain ~15 µg/ml PCPE, was stored at
Recombinant PCPE from conditioned 293 cell media was purified as
described (54) for rPCPE produced in a baculovirus system. The purified
protein, in 20 mM Hepes, 300 mM NaCl pH 7.4, was then concentrated and buffer exchanged, to up to 30 mg/ml in 20 mM Hepes, 150 mM NaCl pH 7.4, using UltraFree
15 microconcentrators (Millipore, 10 kDa cut-off), and stored at
Assay of Enhancing Activity--
Human 3H-labeled
type I procollagen and recombinant BMP-1 with a C-terminal FLAG epitope
were prepared and purified as previously described (23). Procollagen
substrate (400 ng) was incubated at 37 °C either alone or in
combination with 100 ng purified rPCPE and/or 30 ng BMP-1 in 20 µl of
50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2. Following incubation for the
indicated period, reactions were stopped by the addition of 10×
concentrated SDS-PAGE sample buffer containing 2-mercaptoethanol and
boiling for 5 min. Following electrophoresis, processing intermediates
were visualized as previously described (23).
Mass Spectrometry--
Mass spectrometry analysis was performed
on an Applied Biosystems MALDI-TOF Voyager DE-PRO mass spectrometer
operating in delayed extraction and linear mode. The rPCPE sample was
analyzed after dialysis against 0.1 M ammonium acetate and
purification using a ZipTipTM C4 (Millipore).
The matrix was a saturated solution of sinapinic acid (5 mg) in 0.5 ml
of 30% (v/v) CH3CN, 0.1% trifluoroacetic acid.
Analytical Ultracentrifugation--
Sedimentation velocity
experiments were performed using a Beckman XL-I analytical
ultracentrifuge and an AN-60 TI rotor. Experiments were carried out at
20 °C in 10 mM Hepes, 150 mM NaCl, pH 7.4. Two samples of 400 µl at protein concentrations of 0.4 and 1.2 mg/ml
were loaded into 12-mm path cells and centrifuged at 42,000 rpm. Scans
were recorded at 277 nm every 5 min using a 0.03-mm radial spacing.
Sedimentation profiles were analyzed using SEDFIT (55)
(www.analyticalultracentrifugation.com), which takes advantage of a
radial and time-independent noise subtraction procedure (56), to
directly model boundary profiles in terms of a continuous distribution of discrete species and of non-interacting components. This allows the
evaluation of both sedimentation (s) and diffusion
(D) coefficients, from which the molar mass is derived using
the Svedberg equation: M = s
RT/D(1 Rotary Shadowing--
Purified rPCPE was dialyzed against 0.1 M ammonium acetate. Samples were then diluted in the same
buffer and mixed with glycerol (1:1) to obtain final concentrations of
rPCPE ranging from 5 to 15 µg/ml. A drop of the solution was then
placed onto freshly cleaved mica sheets, using the "mica sandwich"
technique (57) and immediately transferred to the holder of a MED 010 evaporator (Balzers). Rotary shadowing was carried out by evaporating
platinum at an angle of 8°, followed by evaporation of carbon at
90°. Replicas were floated onto distilled water, picked up on copper
grids and examined with a Philips CM120 microscope at the "Centre
Technologique des Microstructures" (Université Claude Bernard,
Lyon I).
Small Angle X-ray Scattering--
Synchrotron radiation x-ray
scattering data were collected using standard procedures on the X33
camera (58-60) at the European Molecular Biology Laboratory (EMBL)
Hamburg Outstation on storage ring DORIS III of the Deutsches
Elektronen Synchrotron (DESY), using multiwire proportional chambers
with delay line readout (61). Samples were measured at protein
concentrations of 3.1, 4.6, and 30.8 mg/ml, determined by absorbance at
280 nm using an absorbance of 0.8 at 1 mg/ml calculated from the amino
acid sequence. Scattering curves were recorded at a wavelength (
Data were normalized to the intensity of the incident beam and
corrected for detector response, the scattering of buffer was subtracted, and the difference curves were scaled for concentration using the program PRIMUS.2 To
check for radiation damage and aggregation during the small angle x-ray
scattering (SAXS) experiment, the data were collected in 10 successive
1-min frames. Reduced data sets at low angles were then extrapolated to
zero concentration following standard procedures (62) and then merged
with the higher angle data to yield the final composite scattering curves.
To eliminate scattering from a minor population of high molecular mass
aggregates (which appeared possibly as a result of freezing and/or
concentrating the sample) rPCPE in the low concentration range (3-5
mg/ml) was analyzed directly after further purification by gel
permeation chromatography on Superdex 200, using 20 mM Hepes, 500 mM NaCl, pH 7.4 as elution buffer. Data from
these samples were used for the very low angle region while higher
angle data were obtained with rPCPE purified using the standard
protocol (see above) then adjusted to 500 mM NaCl and
centrifuged for 15 min at 17,500 × g.
The molar mass of rPCPE in solution was calculated by SAXS by comparing
the forward scattering with that from freshly made reference solutions
of bovine serum albumin (molar mass = 66 kDa). The radius of
gyration Rg was evaluated using the Guinier
approximation, Iexp(s) = I
(0)exp(
Low resolution models of the protein were generated ab
initio using the programs DAMMIN (slow mode) (65), DALAI_GA (66), and GASBOR (67), using the smoothed data set generated by GNOM. Both
DAMMIN and DALAI_GA represent the particle as a collection of M Molecular Modeling--
Models were built by homology for the
CUB1, CUB2 and NTR domains with the help of the program Geno3D (68).
This approach first extracts homology-derived spatial constraints on
many atom-atom distances and dihedral angles from the template
structure(s). An alignment is used to determine equivalent residues
between the target and the template. The homology-derived and
stereochemical constraints are then used to generate protein models
that best satisfy these criteria. CUB1 (residues 37-146, numbered from
the start of translation) and CUB2 (residues 159-275) domains were modeled using the acidic seminal fluid protein structure (PDB code:
1SFP) as the template (31), while the template for the NTR module
(residues 318-437) was the structure of TIMP-2 (PDB code: 1BR9)
(51).
Models were fitted by eye to the structures derived from small angle
x-ray scattering using the program MASSHA (69). The program CRYSOL (70)
was then used to calculate the theoretical SAXS profile based on this
three-dimensional arrangement of domains. The program suite CREDO (71)
was used to model the missing portions of the structure corresponding
to the N and C termini (residues 26-36 and 438-449, respectively,
assuming residues 1-25 to represent the signal sequence) and the
CUB1-CUB2 and CUB2-NTR linker regions (residues 147-158 and 276-317, respectively).
Protein Production and Characterization--
In order to produce
sufficient quantities of PCPE for structural analyses, transfected 293 human embryonic kidney cells were employed to establish a clonal cell
line that constitutively produced ~15 µg rPCPE per ml of
conditioned medium. As seen in Fig. 1, although rPCPE had no intrinsic procollagen C-proteinase activity (lanes 1 and 2), it stimulated BMP-1-mediated
processing of type I procollagen to completion under the assay
conditions (lanes 3 and 4). Thus, the rPCPE has
procollagen C-proteinase enhancing activity indicative of a native
conformation suitable for structural analysis.
Analytical Ultracentrifugation--
When subjected to analytical
ultracentrifugation, rPCPE was found to sediment essentially as a
single, slowly sedimenting population with a minor, faster moving
component (Fig. 2). When fitted using a
two component, non interacting species model, the major population
(>95%) gave a sedimentation coefficient
(s20,w) of 3.17 ± 0.02 × 10
While the determination of s20,w by
analytical centrifugation is precise, that for
D20,w is systematically overestimated
even in the case of slight heterogeneity (72). Hence
D20,w was also determined from the
experimentally determined values of
s20,w and molecular mass, determined by mass spectrometry, and the calculated partial specific volume (see
"Experimental Procedures"). This gave a
D20,w value of 5.7 × 10 Rotary Shadowing--
rPCPE molecules were also observed directly
by transmission electron microscopy after rotary shadowing. As shown in
Fig. 3, a mixed population of particles
was observed, about half of which were approximately globular in shape.
The remainder were more rod-like in appearance with approximate
dimensions 80 by 200 Å. A striking feature of most of these rod-like
particles was their dumbbell-like structure consisting of two closely
adjacent lobes (shown circled in Fig. 3).
Small Angle X-ray Scattering--
To get further information on
the shape of rPCPE in solution, the protein was analyzed by SAXS. The
composite experimental scattering curve is shown in Fig.
4. Comparison of the forward scattering
I (0) with that from bovine serum albumin gave an apparent molecular mass for rPCPE of 46 ± 2 kDa. This was consistent with the mass determined by mass spectrometry, or that calculated from protein sequence data, and confirmed that rPCPE was monomeric in
solution.
The radius of gyration Rg of rPCPE, calculated
by Guinier analysis of the data in Fig. 4, was 41 ± 3 Å. Using
GNOM (63), Rg was found to be 43 ± 1 Å.
These values correspond to an equivalent sphere of diameter at least
106 Å, which is larger than that calculated on the basis of the
molecular mass, again showing rPCPE to be an extended molecule. To
determine the maximum dimension of rPCPE from the SAXS data, the pair
distribution function p(r) was also calculated using GNOM.
As shown in Fig. 5, this revealed an
asymmetric curve with a tail extending to a maximum particle length of
~150 Å. In addition, the program ORTOGNOM gave a maximum dimension of 140 ± 10 Å. Thus standard SAXS analysis confirmed the
molecular mass as well as the elongated shape of the protein determined by analytical ultracentrifugation.
To determine the low resolution three-dimensional structure of rPCPE in
solution, the ab initio programs DAMMIN (65), DALAI_GA (66),
and GASBOR (67) were used as described in "Experimental Procedures." The modeling allowed us to neatly fit the experimental data with discrepancy factors Molecular Modeling--
Three-dimensional atomic models of both
CUB domains in rPCPE, as well as the NTR domain, were built on the
basis of sequence homology (Fig. 7) with
domains of known 3D structure. All three models, shown in Fig.
8, A-C, satisfied the
constraints, exhibited good geometry (94, 96, and 95% in favorable
regions of the Ramachandran plot for domains CUB1, CUB2, and NTR,
respectively) and had low energies (
As shown in Fig. 8D, when superimposed on the average
structure generated using DAMMIN, these models fitted well into the overall shape, with room for additional contributions from regions of
unknown structure, especially that of the 42-residue linker region
between CUB2 and NTR. The best fit was obtained, by eye, with the
pointed end corresponding to CUB1 and the blunt end corresponding to
the somewhat larger NTR domain. The asymmetric shapes of the CUB
domains fitted particularly well with the dimensions of the flattened
rod-like structure of the entire PCPE molecule.
The proposed arrangement of domains was well accommodated within the
low resolution shape, but the scattering computed from this model using
the program CRYSOL (70) failed to fit the experimental data (not
shown). The fit was significantly improved however by adding the
missing portions of the structure, corresponding to N-terminal,
C-terminal, and linker regions, using the program suite CREDO (71). The
atomic positions of the domains arranged as in Fig. 8D were
fixed, and additional native-like loops composed of dummy residues were
generated to fit the experimental scattering data. Fig. 8E
presents the best fit conformations of the missing fragments obtained
in five independent runs, all of which, when included with the CUB and
NTR domains, yielded good fits to the experimental data (minimum
Here we have shown that rPCPE is a rod-like molecule. The
elongated shape deduced from analytical ultracentrifugation was confirmed by small angle x-ray scattering, which produced
low-resolution models for the three-dimensional structure of the
molecule. These models were then used as a basis to assemble the
molecule from the predicted structures, built by homology modeling, of
each of its CUB1, CUB2, and NTR domains.
The data obtained in solution are consistent with the images of rPCPE
obtained by transmission electron microscopy after rotary shadowing,
half of which revealed a rod-like structure of dimensions slightly
greater than those determined by SAXS, while the remainder might
correspond to end-on views of the molecule. The frequent observation of
a dumbbell-like substructure by electron microscopy likely corresponds
to the CUB and NTR regions connected by a thin linker region,
consistent with the known susceptibility of this region to proteolytic
attack (73). The lack of a dumbbell-like shape in the models derived
from SAXS probably reflects differences between experimental
techniques: SAXS gives a low resolution structure while rotary
shadowing preferentially coats exposed parts of the structure with a
metal layer up to 20 Å thick. The models shown in Fig. 8E
include relatively thin and extended linkers between the CUB2 and NTR
domains; these are consistent with the rotary shadowing images, though
the linker region would not be as evident at low resolution. Thus the
combination of the different experimental approaches provides a more
complete picture of the overall structure of the PCPE molecule.
The molecular models generated for the CUB and NTR domains fitted well
with the overall shape of rPCPE determined by ab initio modeling from the SAXS data. Furthermore, when the positions and orientations of the domains were manually fitted to the overall shape,
and then the missing portions of the structure (terminal and linker
regions) were modeled using CREDO, it was possible to obtain
satisfactory agreement between observed and theoretical scattering
curves. While this gives strong support to the ab initio modeling, it should be noted that fitting to the SAXS data does not
permit verification of the high resolution structures shown in Fig.
8E. This must await independent structure determination by
x-ray crystallography or NMR.
The elongated nature of the rPCPE molecule is surprising in view of the
calculated isoelectric points of the CUB and NTR domains (pI values of
5.77, 4.33, and 9.21 for CUB1, CUB2, and NTR, respectively) which might
be expected to favor CUB-NTR electrostatic interactions in a more
compact structure. Instead, all three domains are in linear array. This
is the first time that structural information has been obtained on a
molecule containing two adjacent CUB domains. Because of the linear
arrangement, interactions between CUB1 and CUB2 are probably limited to
the external loops, and there is no evidence for stacking of CUB
domains through We have recently shown that PCPE binds more tightly to the intact
procollagen molecule than to its isolated C-propeptide trimer, probably
due to the presence of additional binding sites within the mature
collagen domain (38). Binding of PCPE to sites either side of the
procollagen cleavage site might facilitate a conformational change
leading to stimulation of PCP/BMP-1 activity. Such binding would
involve the CUB domain region of PCPE, which is known to be essential
for PCP/BMP-1 enhancing activity (10). The deduced structure of PCPE is
consistent with such a mechanism, since comparison with the low
resolution structure of the procollagen C-terminal region (75) (Fig.
9) reveals that the CUB domains are far
enough apart to interact with both the C-propeptide and the collagen triple-helix. The future search for putative interaction sites on PCPE
will be aided by the structural information obtained here.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until
further use.
80 °C.
to be 1.005 g/ml, and the solvent
viscosity
to be 1.002 mPa·s, at 20 °C using SEDNTERP software
(V1.01; developed by D. B. Haynes, T. Laue, and J. Philo,
www.bbri.org/RASMB/rasmb.html), which was also used for the calculation
of the corrected s20,w and
D20,w values, and calculated minimum
values of the hydrodynamic diameter Dh.
) of
1.5 Å at a sample-detector distance of 1.8 m covering the
momentum transfer range 0.015 < s < 0.43 Å
1 (s = 4
sin
/
, where 2
is
the scattering angle).
s2Rg2/3),
which is valid for (sRg) < 1.3 (62), and
also from the entire scattering curve using with the indirect transform
package GNOM (63). GNOM was also used to evaluate the forward
scattering I (0) and distance distribution function
p(r). The maximum dimension Dmax was
also estimated using the orthogonal expansion program ORTOGNOM
(64).
1
densely packed beads inside the search volume (a sphere with diameter
Dmax). Each bead belongs either to the particle or to the solvent, and the shape is thus described by a binary string
of length M. Starting from a random string, simulated annealing (DAMMIN) or the genetic algorithm (DALAI_GA) is employed to search for
a compact model that fits the data, after subtracting a small constant
from each data point to force the s
4 decay of
the intensity at higher angles (65, 66). Modeling using DALAI_GA was
carried out using cycles with progressively smaller beads starting at a
radius of 8.5 decreasing to 3.5 Å in steps of 1 Å. The program GASBOR
represents a protein by an assembly of dummy residues (DRs) and uses
simulated annealing to build a locally "chain-compatible" DR-model
inside the same search volume. The DR modeling is able to fit higher
resolution data (without subtraction of a constant) and generally
provides more detailed models than those given by the shape
determination algorithms. With each program, final models were obtained
by averaging at least ten independent runs using the program
DAMAVER.3
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Recombinant PCPE enhances type I procollagen
processing in vitro. Fluorograms are shown of
radiolabeled type I procollagen incubated in the presence or absence of
purified rPCPE and/or BMP-1 for the indicated times. Pro- 1(I) and
pro-
2(I) procollagen chains and pN-
1(I) and pN-
2(I) processing
intermediates generated by BMP-1 activity are indicated by
arrows.
13 s
1 and a translational diffusion
coefficient (D20,w) of 6.5 ± 0.2 × 10
7 cm2 s
1.
Assuming a partial specific volume of 0.721 ml/g (see "Experimental Procedures"), these data correspond to a particle of molecular mass
43 ± 2 kDa. Since this is close to the molecular mass of 48628 Da
determined by mass spectrometry, or that calculated from the amino acid
sequence (45550 Da, glycosylation not included) we conclude that rPCPE
behaves in solution, for the most part, as a monomer.
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Fig. 2.
Analytical ultracentrifugation of rPCPE.
A, snapshots of absorbance as a function of time and
distance from the axis of rotation, showing experimental values
(dots) and theoretical fits (lines).
B, residuals showing differences between observed and
theoretical curves. C, distribution of particles according
to their sedimentation coefficients.
7 cm2 s
1, corresponding to an
hydrodynamic diameter Dh (i.e. that
of a sphere with the same diffusion coefficient) of 74 Å. This result compares with calculated minimum Dh values of 48 and 56 Å for spherical non-hydrated and hydrated (0.4 g of water per
gram of protein) PCPE, respectively. Since the observed
Dh is greater, this indicates that rPCPE has an
elongated structure. The ratio of the observed
Dh to that calculated for spherical, hydrated rPCPE corresponds to a prolate or oblate ellipsoid with an axial ratio
of at least 6.5.
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Fig. 3.
Transmission electron microscopy of rPCPE
after rotary shadowing. Molecules are either globular in shape or
rod-like consisting of two closely adjacent lobes
(circled).
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Fig. 4.
Small angle x-ray scattering curve of rPCPE
in solution. Experimental data (with error bars) are
shown as a function of s (see "Experimental
Procedures"), in comparison with a typical theoretical fit, obtained
here with GASBOR (black line). Fits with DAMMIN and DALAI_GA
were equally good (not shown). Also shown (gray line) is the
best fit curve generated with CREDO corresponding to one of the
structures shown in Fig. 8, including modeled CUB and NTR domains as
well as terminal and linker regions.
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Fig. 5.
Pair distribution function calculated for
rPCPE from the SAXS data. The curve shows the
distribution of interatomic spacings in rPCPE, with a maximum at 150 Å.
(65) of about 0.6. A typical GASBOR
fit is displayed in Fig. 4. With each program, results from at least
ten separate runs were averaged to determine common structural
features. The results are shown in Fig.
6, where each structure is represented by
an array of beads, which serve to define the overall shape. All three
modeling programs gave similar results, where rPCPE appeared as a bent,
rod-like molecule of length ~150 Å. As the rod was somewhat
flattened, the apparent thickness varied depending on the direction of
view. End views showed a maximum width of ~60 Å, consistent with the
highly elongated shape deduced by analytical ultracentrifugation. All
structures showed evidence for both pointed and blunt ends to the rod,
with a pronounced kink nearer the blunt end (Fig. 6).
View larger version (68K):
[in a new window]
Fig. 6.
Structures generated ab initio
from the SAXS data. Molecular shapes are represented in
projection, from three orthogonal directions, by arrays of beads,
generated using the programs DAMMIN (A), DALAI_GA
(B), and GASBOR (C). For each structure, the
results of at least ten independent simulations were averaged.
Orientations with respect to the views shown in the central column (in
which the pointed and blunt ends are to the left and
right, respectively) are indicated by curved
arrows. Structures were aligned using DAMAVER and displayed using
ASSA (76).
3933,
4602, and
4193
kcal/mol, respectively). The compactness of the models, all of which
had the overall form of an oblate ellipsoid, was reasonable. Molecular
dimensions (Fig. 8, A-C) calculated from the models were
globally compatible with those estimated from the SAXS experiments.
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[in a new window]
Fig. 7.
Sequence alignments of PCPE CUB and NTR
domains. CUB domain sequences are aligned with that of the acidic
seminal fluid protein (PDB code: 1SFP) while the NTR sequence is
aligned with that of TIMP-2 (PDB code: 1BR9). Identical residues
(in bold) are indicated by asterisks.
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[in a new window]
Fig. 8.
Three-dimensional models for rPCPE and its
domains. A, CUB1; B, CUB2; and
C, NTR. -helical residues are in red,
-sheets in blue, turns in green, and remaining
structures in white. Disulfide bridges are in
yellow. Dimensions are calculated from the maximum lengths
in projection. Models are displayed as
-carbon traces using
VIEWERLITE (Accelrys). D, superposition of structures shown
in A, B, and C with the averaged
structure generated by DAMMIN from the SAXS data. Three orthogonal
views are shown, represented by beads, with the DAMMIN structure in
semi-transparent gray, the CUB domains in green and the NTR
domain in orange. Structures displayed using ASSA (76).
E, three-dimensional structures of terminal and linker
regions generated using CREDO in five independent runs (shown in
color), together with the domain arrangement from
D shown in white (same scale and same orientation
as in lower left view). Structures displayed as
-carbon traces using MASSHA (69).
= 0.9), as illustrated in Fig. 4. Although the added loops
displayed somewhat different conformations in independent
reconstructions, they were confined in space and permitted one to draw
tentative volumes occupied by the missing portions of the structure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet interactions as observed in the seminal
plasma glycoprotein PSP-I/PSP-II heterodimer (74).
View larger version (49K):
[in a new window]
Fig. 9.
Structural comparison between rPCPE and the
C-terminal region of the procollagen molecule. Both structures are
shown on the same scale. The CUB domains in rPCPE are sufficiently far
apart to interact with both the C-propeptide trimer as well as with a
putative site in the mature collagen domain of the procollagen
molecule.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Becchi for help with the mass spectrometry analysis, E. Kessler for critical reading of the manuscript, and X. Robert for help with the computing.
![]() |
FOOTNOTES |
---|
* This work was supported by the CNRS (Programme Protéomique et Génie des Protéines), the Université Claude Bernard Lyon 1, the Commissariat à l'Energie Atomique, the Association pour la Recherche sur le Cancer, the European Community (Access to Research Infrastructure Action of the Improving Human Potential Programme, Grant HPRI-CT-1999-00017 (to the EMBL Hamburg Outstation); Grant QLK3-2000-00084 (to F. R.)), the International Association for the Promotion of Cooperation with Scientists from the Independent States of the Former Soviet Union, Grants 00-243 and YSF 00-50 (to D. I. S. and M. V. P.), and the National Institutes of Health (Predoctoral Training Grant T32 GM07215 (to B. M. S.) and Grants AR47746 and GM63471 (to D. S. G.)).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.
§ Present address: Sienabiotech, Via Fiorentina 1, 53100 Siena, Italy.
§§ To whom correspondence should be addressed: Institut de Biologie et Chimie des Protéines, CNRS UMR 5086, 7, passage du Vercors, 69367 Lyon Cedex 07, France. Tel.: 33-0-4-72-72-26-67; Fax: 33-0-4-72-72-26-04; E-mail: d.hulmes@ibcp.fr.
Published, JBC Papers in Press, December 15, 2002, DOI 10.1074/jbc.M210857200
2 P. V. Konarev, V. V. Volkov, M. H. J. Koch, and D. I. Svergun, submitted manuscript.
3 V. V. Volkov and D. I. Svergun, submitted manuscript.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PCPE, PCP enhancer; PCP, procollagen C-proteinase; BMP-1, bone morphogenetic protein-1; CUB, module found in complement subcomponents Clr/Cls, Uegf, and BMP-1; mTLD, mammalian tolloid; mTLL, mammalian tolloid-like; NTR, netrin-like; SAXS, small angle X-ray scattering; TIMP, tissue inhibitor of metalloproteinases.
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