NMR Structure of the Netrin-like Domain (NTR) of Human Type I Procollagen C-Proteinase Enhancer Defines Structural Consensus of NTR Domains and Assesses Potential Proteinase Inhibitory Activity and Ligand Binding*

Edvards Liepinsh {ddagger}, László Bányai §, Guido Pintacuda {ddagger} , Mária Trexler §, László Patthy § and Gottfried Otting {ddagger} || **

From the {ddagger}Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17177 Stockholm, Sweden, the §Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1113 Budapest, Hungary, and the ||Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory 0200, Australia

Received for publication, March 18, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Procollagen C-proteinase enhancer (PCOLCE) proteins are extracellular matrix proteins that enhance the activities of procollagen C-proteinases by binding to the C-propeptide of procollagen I. PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain. While the enhancement of proteinase activity can be ascribed solely to the CUB domains, sequence homology of the NTR domain with tissue inhibitors of metalloproteinases suggest proteinase inhibitory activity for the NTR domain. Here we present the three-dimensional structure of the NTR domain of human PCOLCE1 as the first example of a structural domain with the canonical features of an NTR module. The structure rules out a binding mode to metalloproteinases similar to that of tissue inhibitors of metalloproteinases but suggests possible inhibitory function toward specific serine proteinases. Sequence conservation between 13 PCOLCE proteins from different organisms suggests a conserved binding surface for other protein partners.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Procollagen C-proteinase enhancer-1 (PCOLCE1)1 is an extracellular matrix glycoprotein that yields up to 20-fold enhancement of the activities of procollagen C-proteinases such as bone morphogenetic protein-1 (BMP-1) (13), probably mediated by binding to the C-terminal propeptide of type I procollagen (4). Human PCOLCE1 is a 50-kDa protein (55 kDa when glycosylated) (2) of a rod-like shape of about 150 Å length (5). It is built of three structural modules, comprising two CUB domains followed by a C-terminal netrin-like (NTR) domain. The CUB2 and NTR domains are linked by a flexible PEST region of ~40 residues. The enhancement in BMP-1 activity by PCOLCE1 has been assigned to the CUB domains, as the enhancer activity persists after removal of the C-terminal NTR domain by natural processing (2, 3). There is evidence that removal of the NTR domain can substantially increase the enhancer activity (2).

Both PCOLCE1 and its structural and functional homologue PCOLCE2 are collagen-binding proteins, capable of binding at multiple sites on the triple helical portions of fibrillar collagens and also to its isolated C-propeptide trimer (6, 7). This binding activity has been attributed to the CUB domains (5). Much less is known about the function of the NTR domain of PCOLCEs or any other protein. C-terminal fragments of PCOLCE1 comprising the NTR domain have been detected, however, in media conditioned by human brain tumor cells. The fragments, but not full-length PCOLCE1, were shown to be associated with inhibitory activity of matrix metalloproteinase-2 (MMP-2), suggesting that they present a new class of metalloproteinase inhibitors (8).

NTR domains are characterized by a set of conserved disulfide bridges and amino acid sequence homologies. NTR domains have been identified in a wide range of proteins, including the netrin family of proteins, secreted frizzled-related proteins, complement proteins C3, C4, and C5, tissue inhibitors of metalloproteinases (TIMP), and a number of putative Caenorhabditis elegans proteins (9). The putative C. elegans proteins K07C11.3 and K07C11.5 seem to consist only of single, isolated NTR domains, suggesting that NTR domains are stable structural entities. More frequently, however, NTR domains are found as modules in multidomain proteins, where they occur in association with epidermal growth factor-like repeats, CUB domains, fibulin-related domains, cysteine-rich domains related to the Wnt-binding domain of frizzled receptors, and domains homologous to the N-terminal domain VI of laminin B, whey acidic protein domains, follistatin domains, immunoglobulin domains, and Kunitz-type protease inhibitor domains (911).

Based on amino acid sequence homologies, NTR domains present the core structural element of tissue inhibitors of metalloproteinases (TIMP), although the sequence similarities are limited (9). The only structural information about NTR domains to date comes from several TIMP structures and the laminin-binding domain of agrin. The laminin-binding agrin domain is structurally homologous to the NTR subdomain in TIMPs, but its sequence homology to TIMPs and other NTR domains is so low that it escaped identification as an NTR domain before structure determination (12).

Here we report the three-dimensional structure of the NTR domain of PCOLCE1, NTRPCOLCE1, in solution. The NTRPCOLCE1 domain is one of the smallest NTR domains known and, in contrast to TIMPs and the laminin-binding agrin domain, contains the canonical set of three disulfide bonds typical of classical NTR domains (9). The structure establishes a set of characteristic residues that will assist the identification of similar folds from sequence alignments. Functional implications of the fold with regard to potential proteinase inhibitory activity are discussed.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Restriction enzymes were purchased from Promega (Madison, WI) and New England Biolabs (Beverly, MA). DNA polymerase I Klenow fragment, thrombin, and M13 sequencing reagents were from Amersham Biosciences. PCR primers were obtained from Integrated DNA Technologies (Coralville, IA). Ammonium 15N chloride was from Sigma. Human matrix metalloproteinases (MMP-1 proenzyme, active MMP-2, MMP-3 catalytic chain, and active MMP-9) were purchased from Calbiochem; human TACE/ADAM17 was from R & D Systems (Minneapolis, MN). The TACE fluorogenic substrate (DABCYL-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-EDANS) and the MMP fluorogenic substrate (DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2) were from Calbiochem; p-aminophenylmercuric acetate (APMA) was from Sigma. Human plasma kallikrein (Calbiochem) and bovine trypsin (Sigma) were commercial preparations. The synthetic substrates D-Pro-Phe-Arg-pNA and Bz-Phe-Val-Arg-pNA were from Serva (Heidelberg, Germany) and Bachem (Bubendorf, Switzerland), respectively. The pET15b expression vector was from Novagen Merck Kft (Budapest, Hungary). Escherichia coli strains JM109 and BL21(DE3) were from New England Biolabs (Beverly, MA) and Novagen Merck Kft (Budapest, Hungary), respectively. The human liver cDNA library was purchased from Clontech Laboratories, Inc. (Palo Alto, CA).

Cloning of NTRPCOLCE1The DNA segment encoding the NTR domain of human PCOLCE1 was amplified by PCR from human liver cDNA, using the 5'-GCGAATTCATATGATATCTCCTGATGCACCCACCTG-3' sense and 5'-GGCGAAGCTTGGATCCCTACACAGGTTGAGAGGGGCA-3' antisense primers. The amplified DNA was digested with EcoRI and HindIII restriction endonucleases and ligated into M13mp18 Rf digested with the same enzymes. The sequence of the cloned DNA was determined by dideoxy sequencing on both strands. The expression vector pET15b was digested with XhoI, blunt-ended by filling in with DNA polymerase I Klenow fragment, and digested with BamHI restriction endonuclease. The DNA fragment encoding the NTRPCOLCE1 domain was excised from the sequencing vector with EcoRV and BamHI and ligated into the digested vector. E. coli BL21(DE3) cells were transformed with the ligation mixture and plated on 2TY medium containing 100 µg/ml ampicillin. The sequences of the NTRPCOLCE1 inserts of the plasmids were verified by dideoxy sequencing after PCR amplification.

Expression of NTRPCOLCE1Ampicillin-resistant E. coli colonies were screened for protein expression in small volume cultures. Recombinant clones of E. coli were grown in 2-ml aliquots of 2TY medium containing 100 µg/ml ampicillin at 37 °Ctoan A600 of 0.4. Induction was started by addition of isopropyl-{beta}-D-thiogalactopyranoside to a final concentration of 1 mM. The cultures were grown for3hat37 °C, and the expression of recombinant protein was monitored by analyzing the cell lysates with SDS-PAGE. The clone secreting the largest amount of recombinant protein was chosen for large scale protein expression. 3 liters of 2TY medium containing 100 µg/ml ampicillin were inoculated with one colony of the appropriate E. coli clone and grown at 37 °Ctoan A600 = 0.6. Cells were induced by addition of isopropyl-{beta}-D-thiogalactopyranoside to a final concentration of 1 mM and were grown for 3 h at 37 °C.

Isotopic Labeling of NTRPCOLCE1Cells from 100-ml overnight cultures of the appropriate E. coli clone were collected by centrifugation (15 min 5000 x g), and the pellet was suspended in 3000 ml of M9 minimal medium containing 10 mM lactose, 100 µg/ml ampicillin, and 1 g/liter 15NH4Cl and grown at 37 °C to an A600 of about 1.2 to 1.6.

Protein Purification—The cells were harvested by centrifugation with 5000 x g for 15 min, and the pellet was resuspended in 180 ml of 10 mM Tris-HCl, 5 mM MgCl2, pH 8.0. The cells were digested by addition of lysozyme to a final concentration of 0.1 mg/ml at 25 °C for 1 h and subsequently disrupted with an MSE ultrasonicator. Ribonuclease A was added to the suspension to a final concentration of 0.015 mg/ml, and the suspension was incubated for3hat37 °C. The insoluble material (containing the cloned protein in inclusion bodies) was collected by centrifugation (5,000 x g, 15 min) and washed three times with 150 ml of 10 mM Tris-HCl, 5 mM EDTA, 0.05% Triton X-100, pH 8.0 buffer. The pellet was dissolved in 25 ml of 100 mM Tris-HCl, 8 M urea, 5 mM EDTA, 100 mM DTE and was incubated overnight at 25 °C with constant stirring. Insoluble material was removed by centrifugation (5,000 x g, 15 min), and the solubilized proteins were chromatographed on a Sephacryl S300 column equilibrated with 100 mM Tris-HCl, 8 M urea, 10 mM EDTA, 0.1% 2-mercaptoethanol, pH 8.0. Fractions containing the pure protein were identified by SDS-PAGE, pooled, diluted 10-fold with 100 mM Tris-HCl, 8 M urea, 10 mM EDTA, 0.1% 2-mercaptoethanol, and dialyzed extensively against 100 mM ammonium bicarbonate buffer, pH 8.0, at 25 °C. Dialyzed protein was filtered through a membrane (pore size 0.2 µm) and chromatographed on a 10-ml nickel-chelate column. The column was washed first with 100 ml of binding buffer (20 mM Tris, 500 mM NaCl, 5 mM imidazole, pH 7.9), then with 100 ml of wash buffer (20 mM Tris, 500 mM NaCl, 60 mM imidazole, pH 7.9), and finally with 100 ml of elution buffer (20 mM Tris-HCl, 500 mM NaCl, 1 M imidazole, pH 7.9). Fractions of 1 ml were collected and analyzed by SDS-PAGE. Fractions containing pure recombinant NTRPCOLCE1 were pooled, dialyzed against 100 mM ammonium bicarbonate, pH 8.0 buffer, and lyophilized. The lyophilized sample was dissolved in 100 mM ammonium bicarbonate, pH 8.0, chromatographed on a Sephadex G-75 column, and desalted on a Sephadex G-25 column. For enzymatic activity tests, the N-terminal His tag was removed by thrombin digestion. Recombinant protein was dissolved in 5 ml of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) at a final concentration of 1 mg/ml and incubated with 2 units of thrombin at 25 °C for 24 h. The reaction was arrested with 2 mM phenylmethylsulfonyl fluoride, and the digest was applied onto a nickel chelate column. Fractions containing the truncated protein were pooled, dialyzed against 100 mM ammonium bicarbonate buffer at pH 8.0, and lyophilized. The lyophilized sample was dissolved in 100 mM ammonium bicarbonate, pH 8.0, and desalted on a Sephadex G-25 column. The concentration of recombinant NTRPCOLCE1 was determined using a calculated extinction coefficient of 4200 M–1 cm1 at 280 nm. N-terminal sequencing was performed on an Applied Biosystems 471A protein sequencer with an on-line ABI 120A phenylthiohydantoin analyzer. Typical yields before thrombin cleavage were 5 mg of purified NTRPCOLCE1 per liter of cell culture.

Activation of MMP-1 Proenzyme—10 µl of 0.1 µg/µl MMP-1 proenzyme was diluted in 40 µl of 25 mM HEPES, 5 mM CaCl2, 20% glycerol, 0.005% Brij 35, pH 7.5 buffer, and 5 µl of 50 mM APMA dissolved in 0.1 M NaOH was added to the enzyme solution. The mixture was incubated at 37 °C for 3 h.

Fluorescence Measurements—Fluorescence measurements were made with a Fluoromax-3 spectrofluorometer (Jobin Yvon, Edison, NJ) equipped with a thermoregulated sample chamber. The respective excitation (emission) wavelengths in the MMP assays were 280 (360) and 340 (490) nm in the TACE/ADAM17 measurements. 2-nm slits were used for both monochromators. Fluorescence assays were performed in 100 mM Tris-HCl, pH 8.0 (TACE/ADAM17), or 200 mM NaCl, 5 mM NaCl, 0.05% Brij 35, 50 mM Tris-HCl buffers (MMPs) at 25 °C. The concentrations of the enzymes were 2–3 nM, and the thrombin-digested recombinant protein was used at concentrations of 1–15 µM. The concentrations of the fluorogenic substrates of TACE/ADAM17 (DABCYL-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-EDANS) and MMPs (DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2) were 5.3 and 5.6 µM, respectively. The enzyme reactions were followed for 10 min.

Sequence Analyses—The sequences of human (Q15113 [GenBank] ; O14550 [SwissProt] ), mouse (Q61398 [GenBank] ; O35113 [SwissProt] ), and rat (O08628 [GenBank] ) PCOLCE1 sequences, human (Q9UKZ9) and mouse (Q8r4w6) PCOLCE2 sequences, and the sequence of the PCOLCE-related protein of the pufferfish Fugu rubripes (AF016494 [GenBank] ) were from the NCBI data bases. Iterative homology searches with PCOLCE1 and PCOLCE2 as query sequences identified several ESTs corresponding to PCOLCE1 of pig (e.g. BI345440 [GenBank] ); PCOLCE2 of cow (e.g. BE846217 [GenBank] and BE846216 [GenBank] ); PCOLCE1 of the frog Xenopus laevis (e.g. BC046734 [GenBank] ) and PCOLCE2 of the frog Xenopus tropicalis (e.g. AL628110T); and ESTs of PCOLCE-related proteins of zebrafish (e.g. BM316915 [GenBank] , AW232258 [GenBank] ), salmon (e.g. CA055431 [GenBank] , CA050779 [GenBank] ), and rainbow trout (e.g. BX082880 [GenBank] and BX081250 [GenBank] ). Multiple alignments of the amino acid sequences of NTR domains were constructed using ClustalW (13).

Enzyme Assays—MMP-1 proenzyme was activated according to the protocol of the manufacturer as follows. 10 µl of 0.1 µg/µl MMP-1 proenzyme was diluted in 40 µl of 25 mM HEPES, 5 mM CaCl2, 20% glycerol, 0.005% Brij 35 (Merck), pH 7.5 buffer; 5 µl of 50 mM APMA dissolved in 0.1 M NaOH was added to the enzyme solution, and the mixture was incubated at 37 °C for 3 h. The activities of MMP-1, MMP-2, MMP-3, and MMP-9 (2–3 nM) were determined using DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 as substrate (5.6 µM) in 200 mM NaCl, 5 mM NaCl, 0.05% Brij 35, 50 mM Tris-HCl pH 8.0 buffer, at 25 °C. TACE/ADAM17 (2 nM) was assayed with DABCYL-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-EDANS (5.3 µM) in 100 mM Tris-HCl, pH 8.0, at 25 °C. The reaction was followed for 10 min in the Fluoromax-3 spectrofluorometer. The activity of bovine trypsin and human plasma kallikrein on synthetic peptide-pNA substrates was monitored spectrophotometrically using a Cary 300 Scan spectrophotometer. Hydrolysis of peptide-pNA conjugates was monitored at 410 nm, and the initial rates of the reaction were determined. In the case of human plasma kallikrein the enzyme (3 nM) was preincubated for 30 min at 37 °Cin50 mM Tris, 100 mM NaCl, 2 mM CaCl2, 0.01% Triton X-100, pH 7.5 buffer, in the presence of increasing concentrations of thrombin-digested recombinant NTRPCOLCE1 (10–60 µM), and the reactions were initiated by adding the substrate D-Pro-Phe-Arg-pNA at 650 µM final concentration. Bovine trypsin (30 nM) was preincubated for 5 min at 37 °C in 25 mM Tris, 5 mM CaCl2, pH 7.5 buffer, with thrombin-digested recombinant NTRPCOLCE1 (1–10 µM), and the reaction was initiated by adding the substrate Bz-Phe-Val-Arg-pNA at 100 µM final concentration.

NMR Measurements—NMR spectra were recorded at pH 5.5 and 7.5, 28 °C, using ~ 1 mM solutions of the NTRPCOLCE1 construct of Fig. 1A including the His tag. Samples were prepared in 90% H2O/10% D2O or 100% D2O and measured at 1H NMR frequencies of 600 and 800 MHz on Bruker DMX 600 and Varian Unity INOVA 800 NMR spectrometers, respectively. The final structure determination was based on data recorded at pH 5.5. Sequence-specific resonance assignments (Fig. 2) were obtained from double-quantum filtered two-dimensional correlation spectroscopy (DQF-COSY), clean-TOCSY (70 ms mixing time), and NOESY (40 ms and 100 ms mixing time) spectra, recorded with unlabeled protein in H2O and D2O solution at 600 and 800 MHz. In addition, three-dimensional NOESY-15N-HSQC (60-ms mixing time) and TOCSY-15N-HSQC (80-ms mixing time) experiments were recorded in H2O solution on a 15N-labeled protein sample. Most of the NOE restraints were collected from a homonuclear NOESY spectrum recorded at 800 MHz with a sample of unlabeled PCOLCE1 in H2O at pH 5.5 (40-ms mixing time, t1max = 100 ms and t2max = 227.5 ms, 3:9:19 water suppression, 5 days total recording time). The NOE assignments were facilitated by comparison with data recorded at pH 7.5. Stereospecific assignments of C{beta}H2-protons were obtained by a HNHB spectrum. 3J(HN,H{alpha}) couplings were measured by a CT-HMQC-HN experiment (14) as well as by using the program INFIT to fit the line shapes observed in a 15N-HSQC spectrum recorded with a purge-pulse (15). Residual 1H-15N dipolar couplings were measured in a liquid crystal formed with 8% C12E6/n-hexanol (16), using a 15N-HSQC spectrum with {alpha}/{beta} half-filter (17).



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FIG. 1.
Amino acid sequence alignment of the NTRPCOLCE1 module with the laminin-binding domain of agrin, TIMP-2, and other NTRPCOLCE domains. A, alignment with the laminin-binding domain of agrin (residues 1–132) and TIMP-2 (residues 1–127). The alignment is based on the structural alignment provided by DALI (29) and was manually adjusted following visual inspection of superpositions of the structures for the agrin (PDB code 1JC7 [PDB] (12)) and TIMP-2 (PDB code 1BR9 [PDB] (31)) domain, respectively. The sequence of NTRPCOLCE1 shows the construct used for the present structure determination, with residues that are not part of the wild-type sequence shown in italics. The starting residue Pro-25 is residue number 313 in the full-length PCOLCE1 sequence (8). The vertical arrow identifies the location of the thrombin cleavage site. The locations of {alpha}-helices and {beta}-strands are marked by open and filled bars, respectively, above the amino acid sequences. Lines connect Cys residues linked by disulfide bridges. Buried residues, identified by side chain solvent accessibilities below 10%, are highlighted in boldface. Boxes identify residues that are buried in all three proteins. The boxes are filled if, in addition, the residues are consistently uncharged in the 21 different NTR domains aligned by Bányai and Patthy (9). B, alignment of NTR domains from PCOLCE proteins. The sequence numbering is the same as for the human NTRPCOLCE1 domain in A. Filled, crossed, and open boxes indicate, respectively, residues with 5, 10, and 20% side chain solvent accessibility in the NMR structure of the human NTRPCOLCE1 domain. White letters on a black background, white letters on gray background, and black letters on gray background, respectively, identify residues conserved in 100%, 80, and 60% of the aligned sequences. Conserved residues are grouped as follows: F,Y,W; I,L,V,M; R,K; D,N; E,Q; and T,S.

 


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FIG. 2.
15N-HSQC spectrum of the NTRPCOLCE1 module. The spectrum was recorded using a 1 mM sample of uniformly 15N-labeled NTRPCOLCE1 at pH 5.5 and 28 °C at a 1H NMR frequency of 600 MHz. Backbone resonances are assigned with the amino acid types and sequence numbers. Side chain resonances are labeled with lowercase letters.

 

NMR Spectral Evaluation—The NMR data were processed with the program PROSA (18). The cross-peaks in the NOESY spectra were assigned and integrated using the program XEASY (19). 3J(H{alpha},H{beta}) couplings were estimated as 11.0 and 4.0 Hz (±3.0 Hz), respectively, when COSY, TOCSY, and NOESY cross-peaks indicated the presence of large and small couplings, respectively, together with staggered conformations around the C{alpha}-C{beta} bond.

Structure Calculations and Evaluation—The NMR structure was calculated using the program DYANA (20) starting from 50 random conformers. As no long range NOE could be observed for the first 23 residues, only residues 24–154 were included in the structure calculations. The 20 conformers with the lowest residual restraint violations were energy-minimized in water using the program OPAL (21) with standard parameters. The program PALES was used for a best fit of experimental and residual 1H-15N dipolar couplings predicted from the structure (22). The Ramachandran plot was analyzed using PROCHECK-NMR (23). Table I shows an overview of the restraints used and structural statistics. Secondary structure elements and root mean square deviation (r.m.s.d.) values were calculated using the program MOLMOL (24) which was also used to create Figs. 3 and 4. Side chain solvent accessibilities were measured with a spherical probe of 1.4 Å radius and calculated in percent of the accessibilities measured for a fully extended side chain of residue X in a helical Gly-X-Gly peptide (25). The values obtained were averaged over the 20 NMR conformers.


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TABLE I
Structural characteristics for the NMR conformers of the NTRPCOLCEI module

The data pertain to residues 24-154 of the construct shown in Fig. 1A.

 


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FIG. 3.
Solution structure of the NTR module of PCOLCE1 and comparison with the laminin-binding domain of agrin and TIMP-2. A, ribbon representation of the NTRPCOLCE1 module. Disulfide bonds are shown as yellow lines with spheres for the sulfur atoms. The {beta}-strands and {alpha}-helices are numbered as in Fig. 1. White and yellow numbers distinguish strands and helices, respectively. B, ribbon representation of the laminin-binding domain from agrin (PDB code 1JC7 [PDB] ) (12). Secondary structure elements homologous to the NTRPCOLCE1 module are colored as in A. C, ribbon representation of TIMP-2 (PDB code 1BR9 [PDB] ) (30). Same coloring as in A, with the C-terminal segment, which has no counterpart in the NTRPCOLCE1 module, drawn in gray. D, stereo view of the NTRPCOLCE1 module, showing a superposition of the backbone atoms in the 20 conformers representing the NMR structure (Table I), in the same orientation as in A. The disulfide bonds are shown in yellow. Numbers identify sequence positions as in Fig. 1. E, stereo view of the NTRPCOLCE1 conformer closest to the mean structure of the 20 conformers shown in (D), using a heavy atom representation in an orientation rotated by 90o around a horizontal axis. The polypeptide backbone is drawn in green. Backbone carbonyl groups and the flexible C-terminal four residues were omitted. The following colors were used for the side chains: blue, Arg, Lys, His; red, Glu, Asp; yellow, Ala, Cys, Ile, Leu, Met, Phe, Pro, Trp, Val; gray, Asn, Gln, Ser, Thr, Tyr. Heavy lines identify solvent-exposed residues conserved within the groups of mammalian PCOLCE1s, mammalian PCOLCE2s, fish PCOLCEs, and frog PCOLCEs (see text). Selected residues are labeled. Residues from a coherent patch of conserved and solvent-accessible side chains are labeled in red.

 


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FIG. 4.
Comparison of the NTRPCOLCE1 domain with BPTI and PSTI. A, stereo view of backbone traces of the NTRPCOLCE1 domain (red) superimposed onto the trypsin inhibitors BPTI (cyan; PDB code 1BTH [PDB] ) (37) and PSTI (magenta; PDB code 1TGS [PDB] ) (38). The trypsinogen molecule present in the 1TGS [PDB] coordinate set is shown truncated (blue). The superposition of BPTI and PSTI was achieved by superposition of the proteinases in the BPTI·thrombin-E192Q and PSTI·trypsinogen complexes, respectively. The NTRPCOLCE1 domain was superimposed for best fit of the backbone surrounding Lys-32. The arrow identifies the P1 site in BPTI and PSTI. In addition, two selected residues in the NTRPCOLCE1 domain are labeled. B, stereo view of a superposition of the peptide segment 30–36 of the NTRPCOLCE1 domain (using the conformer closest to the mean structure in this segment; backbone in red), the proteinase-binding peptide segment 13–19 of BPTI (backbone in cyan), and the proteinase-binding peptide segment 16–22 of PSTI (backbone in magenta). The C{alpha} atoms of the residues in the P1–P3 and P1'–P3' sites of the inhibitors are identified. Spheres mark the N- and C-atoms of the N- and C-terminal ends, respectively, of the polypeptide segments. The following colors were used for the side chains: blue, Arg, Lys; yellow, Ala, Cys, Ile, Pro; gray, Asn, Gln, Thr, Tyr. C, sequence alignment of the inhibitor segments shown in B. Boxes identify residues with closely superimposable backbones.

 


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NMR Spectra—The line widths observed in the NMR spectra of the NTRPCOLCE1 domain were characteristic of a monomeric protein (Fig. 2). Particularly narrow resonances were observed for the N-terminal 24 and the C-terminal 4 residues, indicating increased mobility for these polypeptide segments on a nanosecond time scale. Pro-25 and Pro-150 thus mark the boundaries of the structured domain. The amide proton resonances of the N-terminal 11 residues and of His-20 could not be observed, presumably because of exchange broadening. No NOEs could be observed for the side chain NH2 resonances of Gln-33, Gln-100, and Gln-152, prohibiting their assignment. The protein contains 17 proline residues. Because of spectral overlap, only incomplete resonance assignments were obtained for the proline residues 25, 86, 103, 125, and 126. Pro-102 is the only residue for which a cis-peptide bond was identified. Besides Pro-126, this is the only proline residue which is totally conserved in all known PCOLCE proteins (Fig. 1B).

Structural Statistics—The number of restraints and the final r.m.s.d. values are characteristic of a well determined structure. No residue was found in the forbidden region of the Ramachandran plot (Table I). The relative orientation of the regular secondary structure elements was verified by 57 residual dipolar 1H-15N coupling constants that were not used as restraints in the structure calculation; a best fit of the experimental values with those predicted from the structure yielded a correlation coefficient of 0.9.

Structure of the NTRPCOLCE1 Module—The fold of NTRPCOLCE1 consists of a {beta}-barrel flanked by {alpha}-helices at the N-and C-terminal ends (Fig. 3A). Regular secondary structure elements include residues 40–46 ({alpha}1), 49–60 ({beta}1), 66–70 ({beta}2), 74–78 ({beta}3), 92–96 ({beta}4), 109–117 ({beta}5), 121–123 ({beta}6), 129–132 ({beta}7), and 135–146 ({alpha}2) as identified by the Kabsch-Sander algorithm (26) in the majority of the NMR conformers. The strands {beta}1 and {beta}4 contain {beta}-bulges at Val-55 and Lys-56 and at Tyr-76, respectively. The disulfide bonds agree with the prediction (9) and are consistent with those determined for the homologous NTR module of the secreted Frizzled-related protein-1 (27).

Buried Side Chains—All side chains with less than 10% side chain solvent accessibility are hydrophobic or uncharged in NTRPCOLCE1 (Fig. 1A). Structural conservation between the NTR domains from human PCOLCE1 and other PCOLCE NTR domains is indicated by about 35% sequence identity (6, 28) but also by the fact that the buried side chains in NTRPCOLCE1 (Fig. 1B) are almost always hydrophobic or uncharged also in the other PCOLCE proteins (Fig. 1B).

Comparison with Agrin and TIMP-2—A search of the Protein Data Bank for structurally related proteins with the program DALI (29) yielded the laminin-binding domain of agrin (12) and the metalloproteinase-2 inhibitor (TIMP-2) (30) as the proteins with the highest Z-scores, with backbone r.m.s.d. valves to the NTRPCOLCE1 module of 2.7 and 2.5 Å, respectively, for 110 aligned residues. Despite high Z-scores (11.9 and 9.7, respectively), the sequence identities between NTRPCOLCE1, TIMP-2, and the agrin domain are below 10%. Both the agrin domain and TIMP-2 have several insertions compared with NTRPCOLCE1, including additional helices. Furthermore, the sequence alignment based on structural homology shows that the locations of most of the disulfide bonds are not very well conserved (Fig. 1A), although the disulfide bonding pattern has been used successfully to identify the sequence homology between different NTR modules (9). In particular, the second disulfide bond in TIMP-2 is formed between different polypeptide segments (Figs. 1A, 3A, and 3C). Although the structural alignment shown in Fig. 1 mostly confirms previous sequence alignments (5, 9), it differs in important details, as expected for proteins of low sequence homologies. For example, the strand {beta}4, which contains three buried, and hence structurally important, residues in a row, has not been aligned correctly before.

Functional Studies—The structural similarity of the NTRPCOLCE1 domain with TIMP proteins suggests that it could act as a proteinase inhibitor. Even at micromolar concentrations, however, we could not detect any significant inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9, tumor necrosis factor-{alpha}-converting enzyme (TACE), trypsin, plasma kallikrein, or thrombin with our construct, neither with the full-length construct nor after cleavage of the N-terminal His tag with thrombin (cleavage site shown in Fig. 1A).

As C-terminal fragments of PCOLCE1 containing the NTR module have been reported to inhibit MMP-2 (8), we attempted to model a NTRPCOLCE1·MMP complex by superimposition of the {beta}-barrel of the NTRPCOLCE1 structure onto the {beta}-barrels of the TIMP structures in the MMP-3·TIMP-1 and MMP-1·TIMP-2 complexes (31, 32). This simple model resulted in numerous clashes between the backbones at the active site of the MMPs that could not be relaxed in any obvious way, resulted in a far smaller interaction surface than in the MMP·TIMP complexes, and buried a charged residue (Lys-32) in the middle of the interface. Clearly, any inhibitory complex between NTRPCOLCE1 and MMPs would have to use a different binding mode.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The NTRPCOLCE1 domain is the first structure of a classical NTR module. Despite low sequence identities, its structural fold is closely related to the {beta}-barrel part of TIMPs, raising the possibility that it functions as a metalloproteinase inhibitor (8, 9). MMP inhibition by TIMPs depends on a conserved N-terminal Cys residue in the TIMP proteins that coordinates one of the zinc atoms of metalloproteinases via the N-terminal NH2 and the first backbone-carbonyl group. Crystal structures of complexes with MMPs showed that this residue becomes completely buried during complex formation, i.e. a longer N-terminal segment could not be accommodated (31, 32). In agreement with this observation, no proteinase-inhibitory activity has been reported to date for the laminin-binding agrin domain, although it contains only a single additional residue preceding the first disulfide-bonded Cys residue (Fig. 1A) (12). In contrast to TIMPs and the laminin-binding agrin domain, the NTRPCOLCE module is found at the C-terminal end of multidomain proteins rather than at their N terminus, implying that TIMP·MMP complexes are not good models for its interaction with targets.

Nonetheless, C-terminal PCOLCE1 fragments comprising the NTR domain (referred to as CT-PCPE), but not full-length PCOLCE1, have been shown to inhibit MMP-2 with an IC50 value of about 0.5 µM, i.e. a value several hundredfold higher than for TIMP/MMP interactions (8). These data contrast our result that NTRPCOLCE1 does not have any inhibitory effect on MMP-2 at concentrations of up to 10 µM.

The discrepancy could be explained by the different constructs used. Compared with the NTRPCOLCE1 construct used in the present study, CT-PCPE had a heterogeneous N terminus and included between 9 and 25 additional residues from the wild-type sequence preceding Pro-25, with shorter CT-PCPE species showing increased inhibitory activity (8). It seems unlikely that the additional N-terminal residues could affect the structure of the NTR module, as they are almost certainly flexible. These residues are located in the linker region between the second CUB domain and the NTR domain in the full-length PCOLCE1 protein. The linker is rich in proline, glutamate, serine, and threonine residues, which is a characteristic feature of unstructured proteins (33). Flexibility of this linker has also been inferred from electron microscopy data (5) and susceptibility to proteolytic attack (34). Although the linker residues may still be important for binding to MMP-2, the relatively high IC50 value reported for CT-PCPE suggests that MMP-2 is not the main target of the NTR domain of PCOLCE1 (8).

Although TIMPs inhibit members of the matrixin family of the metzincin superfamily, PCOLCE1 and PCOLCE2 interact with BMP-1 which is a member of the astacin family of the same superfamily. It has thus been speculated that inhibition of a metalloproteinase is one of the main functions of the NTRPCOLCE module, in particular as other proteins containing NTR modules are also involved in proteolytically regulated pathways (911).

Based on model building studies, BMP-1 seems unlikely to form a classical protease-inhibitor complex with the NTRPCOLCE module. The structure of BMP-1 has not been determined, but coordinates are available for the homologous proteinase astacin (PDB code 1AST [PDB] ) (35) which shows a much deeper active-site cleft than other metalloproteinases. Unless the cleft is significantly more open in BMP-1, an NTRPCOLCE domain could hardly contact the active-site zinc of the proteinase.

TACE is a member of the ADAM family of metalloproteinases that have a more shallow active-site cleft (36). Experimentally, however, our NTRPCOLCE1 construct did not show an inhibitory effect for this proteinase.

Inhibitory polypeptide segments binding to the active-site cleft of proteinases often are highly solvent-exposed in their uncomplexed state, form irregular secondary structure, and contain cystine residues that stabilize their structure by linkage to the core of the proteinase inhibitor. Visual inspection of the structure of the NTRPCOLCE1 domain shows that all these criteria are fulfilled by the N-terminal segment that is connected by two disulfide bonds to the loop between strands {beta}4 and {beta}5 (Figs. 1A and 3A). Moreover, comparison of this segment with the inhibitory polypeptide segments of basic pancreatic trypsin inhibitor (BPTI) and porcine secretory trypsin inhibitor (PSTI) shows remarkably close structural similarities, when the specificity-determining P1 site lysine residues of BPTI and PSTI are superimposed with Lys-32 of the NTRPCOLCE1 domain.

The BPTI and PSTI coordinates of Fig. 4A were taken from co-crystal structures with thrombin-E192Q and trypsinogen, respectively (37, 38). These proteinases have very similar structures and can be precisely superimposed, highlighting differences and similarities in the binding modes of BPTI and PSTI. BPTI and PSTI have very different folds and make different contacts with the proteinase at the fringes of the binding site. Fig. 4B shows that the backbone and C{beta} atoms of the sites P2 to P2' of BPT1 and PST1 can be superimposed with residues 31 to 34 of the NTRPCOLCE1 domain. Overall, the NTRPCOLCE1 domain matches PSTI better than BPTI, as five rather than four residues superimpose with PSTI, and three of them have identical side chains. In addition, although BPTI contacts the proteinase with the entire segment comprising residues 13–19, Tyr-20 in PSTI is the last residue in intimate contact with trypsinogen. The different backbone conformation of the NTRPCOLCE1 domain after Cys-34 (Fig. 4, B and C) could thus be accommodated better in a PSTI-like complex. Although the four residues comprising the segment between Cys-30 and Gln-33 are totally conserved between NTRPCOLCE1 from human, pig, mouse, and rat, Cys-34 is replaced by Tyr in the mouse and rat proteins (Fig. 1B), further increasing the sequence homology with PSTI (Fig. 4C).

No inhibitory activity of the NTRPCOLCE1 domain against plasmin was reported (8), nor could we detect any inhibitory activity against trypsin, thrombin, or plasma kallikrein. This may be explained by the loop regions around Glu-64 and Gln-100 that approach the protease more closely (Fig. 4A). It is conceivable that a not very different serine protease could present the correctly matching surface shape for binding. Whereas BPTI and PSTI inhibit a wide range of proteinases, the additional loop regions present in the NTRPCOLCE1 domain would provide a mechanism for enhanced specificity in target recognition.

The putative proteinase-binding peptide segment is conserved between mammalian PCOLCE1 sequences but is different for the NTR domain of the less widely expressed PCOLCE2 protein (Fig. 1B). If our model is correct, the NTR domain of PCOLCE2 would not inhibit the same proteinase as PCOLCE1 or would do so with different efficiency. Whereas PCOLCE1 is expressed at high levels in the submucosal layer of the gut, PCOLCE2 is primarily expressed in nonossified cartilage in developing tissues (6), suggesting that protease inhibition may not be a similarly important function for PCOLCE2. Due to a 23-residue deletion in the N-terminal segment of its NTR module, C-terminal fragments of human PCOLCE2 are also unlikely to inhibit MMP-2 in the same way as CT-PCPE.

The amino acid sequence of the active-site binding loop of ovomucoid third domains, which are Kazal-type serine proteinase inhibitors like PSTI, has been shown to be hypervariable between different bird species (39). The residues in the putative active-site binding loop of the NTR domains of PCOLCEs appear to be more highly conserved. Thus, the sequence CPKQ is found at the N terminus of the NTRPCOLCE1 domain in all mammalian species, although the sequence CQQK is characteristic of NTRPCOLCE2 domains (Fig. 1B). There is evidence for only a single type of PCOLCE protein in fish, where the N-terminal sequence is CAKA with high conservation. It appears as if the distinction between PCOLCE1 and PCOLCE2 occurred late in evolution which is also apparent from a comparison of the entire NTR domain sequences. This result is also reflected in the frog NTRPCOLCE1 sequence that is much closer to NTRPCOLCE2 sequences than the other NTRPCOLCE1 domains (Fig. 1B). The residues of any target protein that interact with the N-terminal segments of the NTR domains of PCOLCEs might thus be expected to be conserved within mammals, fish, and amphibians but not between them.

Only few residues are totally conserved between all NTR domains of PCOLCE proteins, and most of them are buried inside the protein, suggesting structural importance (Fig. 1B). In order to identify a potential surface site for intermolecular binding to another protein, we assumed that the binding partner is conserved within the groups of PCOLCE1, PCOLCE2, and fish PCOLCE proteins but not between those groups. To account for the evolutionarily intermediate position of the frog PCOLCE proteins, a residue was considered conserved if the sequence identities within the NTRPCOLCE1 or NTRPCOLCE2 domains extended to at least one of the corresponding frog sequences. The graphical display of the result (Fig. 3E) was further simplified by disregarding the conserved disulfide bonds and buried residues with less than 5% side chain solvent accessibility. This procedure identified an extended patch of conserved residues with uncharged residues in the center and positive charges at the rim. In particular, Arg-36 is highly conserved (Fig. 1B). Arginine residues are particularly frequently involved in intermolecular protein-protein interactions (40). We propose that this patch marks a conserved interaction site with a binding partner.

The NMR structure of the human NTRPCOLCE1 domain presents a blueprint for the identification and modeling of other NTR domains. The NTRPCOLCE1 domain belongs to the smallest of the NTR domains identified to date. Hence, residues buried in the NTRPCOLCE1 domain are likely to be structural prerequisites of NTR domains in general. The set of consistently buried and uncharged residues identified in the structural alignment of Fig. 1A thus provides a fingerprint for the identification of novel NTR domains and improved sequence alignments that is independent of the pattern of disulfide bonds. Whereas NTR domains usually form at least two, but mostly three disulfide bonds (9), the structure of the laminin-binding domain of agrin shows that, in a somewhat larger protein, a single disulfide bond can suffice for a stable NTR fold (Fig. 1). Structural stability combined with significant spatial separation between the N and C termini endow the NTR domain with the classical features of a structural module in multimodular proteins.


    FOOTNOTES
 
The atomic coordinates and structure factors (code 1UAP) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The NMR chemical shifts have been deposited at the BioMagResBank under code BMRB 5751.

* This work was supported by a grant from the Swedish (Vetenskapsråd) and the Australian Research Councils, by Grant NKFP1/044/2001 from the Hungarian National Research and Development Program, and Grant OTKA/T034317 from the Hungarian Research Fund, Budapest, Hungary. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Recipient of a Postdoctoral Fellowship by the European Union Contract HPRN-CT-2000-00092. Back

** Federation Fellow of the Australian Research Council. To whom correspondence should be addressed: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. Tel.: 61-2-6125-6507; Fax: 61-2-6125-0750; E-mail: gottfried.otting{at}anu.edu.au.

1 The abbreviations used are: PCOLCE, procollagen C-proteinase enhancer protein; APMA, p-aminophenylmercuric acetate; BMP-1, bone morphogenetic protein-1; BPTI, basic pancreatic trypsin inhibitor; CT-PCPE, C-terminal domain of procollagen C-terminal proteinase enhancer; CUB domain, complement-Uegf-BMP-1 domain; DABCYL, 4-{[(4-dimethylamino)phenyl]azo}benzoic acid; DNP, 2,4-dinitrophenyl; DTE, 1,4-dithioerythriol; EDANS, 5-[(2'-aminoethyl)-amino]naphthalenesulfonic acid; EST, expressed sequence tag; HSQC, heteronuclear single quantum coherence; MMP, matrix metalloproteinase; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; NTR domain, netrin-like domain; PDB, protein data bank; PEST region, region rich in Pro, Glu, Ser, and Thr residues; PSTI, pancreatic secretory trypsin inhibitor; r.m.s.d., root mean square deviation; TACE/ADAM17, tumor necrosis factor-{alpha} converting enzyme/A disintegrin and metalloproteinase domain 17; TIMP, tissue inhibitor of metalloproteinase; TOCSY, total correlation spectroscopy; pNA, p-nitroanilide. Back



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