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• W2103230402 abstract "The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule. The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule. Minicollagens of nematocysts in Hydra, corals, and other cnidaria are very unusual proteins with structural properties not shared by other invertebrate or vertebrate collagens. From both the N and C terminus of the collagen triple helix emerge three polyproline-II-type helices, which consist of 5–23 proline or hydroxyproline residues (1Vollmer S.V. Palumbi S.R. Science. 2002; 296: 2023-2025Crossref PubMed Scopus (213) Google Scholar, 2Wang W. Omori M. Hayashibara T. Shimoike K. Hatta M. Sugiyama T. Fujisawa T. Gene (Amst.). 1995; 152: 195-200Crossref PubMed Scopus (23) Google Scholar, 3Kurz E.M. Holstein T.W. Petri B.M. Engel J. David C.N. J. Cell Biol. 1991; 115: 1159-1169Crossref PubMed Scopus (99) Google Scholar). Each of the polyproline-II-type helices is terminated by a small Cys-rich domain termed minicollagen Cys-rich domain (MCRD). 1The abbreviations used are: MCRD, minicollagen Cys-rich domain; NOWA, nematocyst outer wall antigen; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetremethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; HPLC, high pressure liquid chromatography; Boc, t-butoxycarbonyl; EGF, epidermal growth factor.1The abbreviations used are: MCRD, minicollagen Cys-rich domain; NOWA, nematocyst outer wall antigen; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetremethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; HPLC, high pressure liquid chromatography; Boc, t-butoxycarbonyl; EGF, epidermal growth factor. The N- and C-terminal MCRDs are homologous and share the cysteine pattern CXXXCXXXCXXXCXXXCC. A small propeptide region preceding the N-terminal MCRD is cleaved off during expression, and mature minicollagen has a rather symmetrical appearance with closely similar structural elements at both sides. This bipolar nature of minicollagen is unique among all other known collagens (4Bateman J.F. Lamande S.R. Ramshaw J.A.M. Comper W.D. Collagen Superfamily. Extracellular Matrix. Molecular Components and Interactions. 2. Harwood Academic Publisher, Amsterdam1996Google Scholar, 5Engel J. Science. 1997; 277: 1785-1786Crossref PubMed Scopus (60) Google Scholar, 6van der Rest M. Garrone R. FASEB J. 1991; 5: 2814-2823Crossref PubMed Scopus (958) Google Scholar, 7Ricard-Blum S. Dublet B. van der Rest M. Unconventional Collagens. Oxford University Press, New York2000Google Scholar) and suggests a special function. A unique function of minicollagen is also suggested by its restricted appearance in the capsule wall of nematocysts. Nematocysts are complex explosive organelles, which basically consist of a capsule, an inverted tubule armed with spines, and an operculum. The tubule is connected to the capsule wall and is twisted in many turns inside the osmotically charged capsule matrix. Following stimulation, the internal tube is expelled, the osmotic pressure is released, and the capsule contents including toxins are released at the tubule end. This specialized form of exocytosis proceeds with ultrafast rates and accelerations comparable to those of a fired bullet (8Holstein T. Tardent P. Science. 1984; 223: 830-833Crossref PubMed Scopus (189) Google Scholar). The capsule wall resists more than 150 atmospheres of osmotic pressure in the charged state. For Hydra nematocysts it consists mainly of two proteins, minicollagen and nematocyst outer wall antigen (NOWA) (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar, 10Özbek S. Pertz O. Schwager M. Lustig A. Holstein T. Engel J. J. Biol. Chem. 2002; 277: 49200-49204Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 11Engel U. Özbek S. Engel R. Petri B. Lottspeich F. Holstein T.W. J. Cell Sci. 2002; 115Crossref PubMed Scopus (62) Google Scholar). The two proteins can be dissolved from capsule preparations only under reducing conditions. Already at a time when the amino acid sequence of minicollagens was unknown it was found that these collagens formed disulfide cross-linked polymers that were insoluble in SDS but easily soluble in the presence of a reducing agent (12Lenhoff H.M. Kline E.S. Hurley R. Biochim. Biophys. Acta. 1957; 26: 204-205Crossref PubMed Scopus (25) Google Scholar, 13Blanquet R. Lenhoff H.M. Science. 1966; 154: 152-153Crossref PubMed Scopus (42) Google Scholar). Following the discovery of a family of minicollagens (3Kurz E.M. Holstein T.W. Petri B.M. Engel J. David C.N. J. Cell Biol. 1991; 115: 1159-1169Crossref PubMed Scopus (99) Google Scholar) and recombinant expression of minicollagen-1 (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar) it was found that the proteins are expressed in a soluble precursor form present in the endoplasmic reticulum and post-Golgi vacuoles in Hydra. They are converted to the disulfide-linked assembly form of the nematocyst wall upon wall compaction, during which a dense and well defined capsule wall is formed (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar). The morphological changes, a loss of accessibility to antibodies against minicollagen-1, and a parallel loss of solubility under non-reducing conditions suggested a close link between disulfide polymerization and the condensation of wall proteins. Both processes provide an explanation for the unusually high tensile strength of the mature nematocyst wall. Minicollagen-1 of Hydra recombinantly expressed in mammalian cells contains internal disulfide bonds in its MCRDs but no interchain disulfide cross-links between chains (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar). The trimeric collagen molecules dissociated into single chains when heated to 45 °C under non-reducing conditions (10Özbek S. Pertz O. Schwager M. Lustig A. Holstein T. Engel J. J. Biol. Chem. 2002; 277: 49200-49204Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Reduction did not influence this transition temperature, indicating that only the collagen domain is responsible for trimerization. Recombinant minicollagen-1 was found to form aggregates in electron micrographs but no disulfide bridges were formed spontaneously under in vitro conditions (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar). This material behaved like the precursor form in vivo and could be solubilized by SDS or other denaturants under non-reducing conditions. It was therefore concluded that disulfide isomerases or other external parameters are required for a disulfide reshuffling process by which internal disulfide bridges are converted to intermolecular links (14Özbek S. Engel U. Engel J. J. Struct. Biol. 2002; 137: 11-14Crossref PubMed Scopus (21) Google Scholar). More recent results suggest that the structural function of minicollagens in wall hardening is complemented by NOWA (11Engel U. Özbek S. Engel R. Petri B. Lottspeich F. Holstein T.W. J. Cell Sci. 2002; 115Crossref PubMed Scopus (62) Google Scholar). Altogether 10 domains were identified in this protein, namely a SCP-domain (see Smart data base, smart.embl-heidelberg.de, smart00198), a C-type lectin domain (CTLD, smart00034), and eight C-terminal domains with homology to the MCRDs. In particular, the pattern of six cysteines is shared by the corresponding domains in NOWA and minicollagen. The presence of homologous Cys-rich domains in both proteins suggested a joint function in disulfide-mediated polymerization. Supportive evidence was obtained by the analysis of breakdown products of native nematocyst capsules after limited sonification without reduction. 2S. Özbek, E. Pokidysheva, M. Schwager, T. Schulthess, N. Tariq, D. Barth, A. G. Milbradt, L. Moroder, J. Engel, and T. W. Holstein, manuscript in preparation.2S. Özbek, E. Pokidysheva, M. Schwager, T. Schulthess, N. Tariq, D. Barth, A. G. Milbradt, L. Moroder, J. Engel, and T. W. Holstein, manuscript in preparation. Minicollagen and NOWA formed disulfide cross-linked units that, like the entire capsule, were readily dissolved under even mild reducing conditions. Disulfide reshuffling processes and the formation of disulfide-linked complexes are common mechanisms in the extracellular space, and some recently explored systems may be referenced (15Mullen G.E. Kennedy M.N. Visintin A. Mazzoni A. Leifer C.A. Davies D.R. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3919-3924Crossref PubMed Scopus (64) Google Scholar, 16Wieringa R. De Vries A.A. Post S.M. Rottier P.J. J. Virol. 2003; 77: 12996-13004Crossref PubMed Scopus (34) Google Scholar, 17Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Crossref PubMed Scopus (453) Google Scholar, 18Wagner D.D. Lawrence S.O. Ohlsson-Wilhelm B.M. Fay P.J. Marder V.J. Blood. 1987; 69: 27-32Crossref PubMed Google Scholar, 19Li P.P. Nakanishi A. Clark S.W. Kasamatsu H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1353-1358Crossref PubMed Scopus (58) Google Scholar). The minicollagen/NOWA system may stand as a prototype for the controlled formation of a highly stable matrix layer by disulfide linkage. To understand the assembly mechanism, the three-dimensional structure of the MCRDs involved is needed. The core of the MCRD consists of only 20 residues with 6 closely positioned Cys residues. At the start of this study it was not known whether the very small domains were autonomous or stable but only as part of a larger structure. The disulfide connections were also not known and could not be explored by limited proteolysis because of the lack of suitable cleavage sites. These problems were approached by solving the structure of the C-terminal Cys-rich domain of minicollagen-1 by NMR spectroscopy. A new fold was found, with disulfide connections between Cys2 and Cys18, Cys6 and Cys14, and Cys10 and Cys19. Oxidative folding occurs at high rates in the presence of an oxidized glutathione/reduced glutathione redox buffer with formation of almost exclusively one single isomer. A highly conserved Pro residue located between Cys10 and Cys14 induces and probably directs correct disulfide connections. The presented structure proposes likely candidates for disulfide reshuffling supposed to be a key reaction during nematocyst morphogenesis. Preparation 1—The 6xStBu protected peptide Ac-PCPPVCVAQCVPTCPQYCCPAKRK-NH2 was synthesized on Fmoc-Rink-Amide polyethylene glycol-dimethylacrylamide copolymer resin by the Fmoc/tBu strategy using double couplings with Fmoc-Xaa-OH/HBTU/HOBt/DIEA (4:4:4:8), intermediate Fmoc cleavage with 20% piperidine in N,N-dimethylformamide, and acetic anhydride/DIEA (4:8) for N-terminal acetylation. After resin cleavage/deprotection with trifluoroacetic acid/phenol/H2O/thioanisole/1,2-ethanedithiol (82.5:5:5:5:2.5) the product was isolated by reverse phase-HPLC: yield = 6%; HPLC: tR = 12.5 min (>98%); electrospray ionization-mass spectrometry: m/z = 1055.0 [M+3H]3+; 1581.8 [M+2H]2+; Mr = 3162.31 calculated for C136H230N32O29S12. The Cys-protecting groups were cleaved in trifluoroethanol/H2O with tributylphosphine (60 eq) at room temperature for 5 h, and the resulting fully deprotected peptide was oxidized at pH 8.0 in the presence of GSSG/GSH (9 eq, 10:1) under air atmosphere at 7 °C. The crude product was purified by preparative size exclusion column chromatography, yield: 20%; HPLC: tR = 12.3 min (>98%); electrospray ionization-mass spectrometry: m/z = 1314.8 [M+2H]2+, 876.4 [M+3H]3+; Mr = 2627.23 calculated for C112H176N32O29S6. Preparation 2—The linear peptide was synthesized with an ABI 433A synthesizer. Couplings were carried out on a Fmoc-Lys(Boc)-polyethylene glycol-polystyrene resin (Perseptive Biosystems, 0.21 mmol/g) using Fmoc amino acids (Fmoc-Arg(Pbf), Fmoc-Lys(Boc), Fmoc-Tyr(tBu), Fmoc-Cys(trityl), and Fmoc-Gln(trityl)) (Anaspec). HATU (O-(7-azabenzotriazol-1-yl)-1.1.3.3-tetramethyluronium hexafluorophosphate, (Perseptive Biosystems) (4.0 eq.)/diisopropylethylamine mediated peptide couplings. The peptide was cleaved from the resin with trifluoroacetic acid containing thioanisole (5% v/v), ethanedithiol (3% v/v), and anisole (2% v/v). After precipitation in diethylether, the crude peptide was purified by preparative HPLC (Vydac® C18, 10–15 μm, 300 Å, 250 × 50 mm, W.R. Grace, Columbia, MD). The peptide was characterized by electrospray ionization/quadrupole/time-of-flight mass spectrometry and amino acid analysis. A mass of 2590.9 Da was determined, and the expected amino acid composition was found. The lyophilized purified peptide was dissolved in 25 mm Tris/HCl, pH 6.0, containing 100 mm NaCl at a concentration of 0.02 mm. Folding was allowed to proceed under N2 for 72–96hat4 °C. Oxidation was induced by changing the pH to 8.0–8.1 with saturated Tris and the addition of reduced and oxidized glutathione (10:1 molar ratio) to a final concentration of 1 mm. The solution was exposed to air at 4 °C for 4–10 days. Oxidation was stopped by the addition of trifluoroacetic acid to give a pH of 1.3. The oxidized peptide was purified by preparative HPLC. The yield of oxidized peptide was 103 mg (16% of theoretical yield). Mass spectroscopy of the oxidized peptide showed a molecular mass of 2585.1 Da. Structure 1—NMR experiments for conformational analysis were carried out at 283 K on Bruker DRX500, DMX750, and DX900 spectrometers using a 3 mm sample of the peptide dissolved in a H2O/D2O (9:1) mixture at pH 3.5. Resonance assignments were performed according to the method of Wüthrich (38Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). 173 experimental interproton distance constraints were extracted from two-dimensional-nuclear Overhauser effect spectroscopy (39Jeener J. Meier B.H. Bachman P. Ernst R.R.J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4805) Google Scholar) experiments with mixing times between 75 and 200 ms. Five hydrogen bonds were identified from temperature shifts and 1H/2H exchange. Acceptor carbonyl groups were identified in initial structure calculations. The nuclear Overhauser effect intensities were converted into interproton distance constraints using the classifications of very strong, 1.7–2.3 Å; strong, 2.2–2.8 Å; medium, 2.6–3.4 Å; weak, 3.0–4.0 Å; very weak, 3.2–4.8 Å, and the distances of pseudo atoms were corrected as described by Wüthrich (38Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Distance geometry and molecular dynamics-simulated annealing calculations were performed with the INSIGHTII 98.0 software package (Accelrys, San Diego, CA) on Silicon Graphics O2 R5000 computers (SGI, Mountain View, CA) as described recently (25Milbradt A.G. Kerek F. Moroder L. Renner C. Biochemistry. 2003; 42: 2404-2411Crossref PubMed Scopus (32) Google Scholar). In brief, one hundred structures were generated by distance geometry and refined with molecular dynamics-simulated annealing steps. The experimental constraints were applied at every stage of the calculations. The coordinates and structural restraints have been deposited in the Brookhaven Protein Data Bank under accession number 1SOP. Structure 2—The chemically synthesized C-terminal MCRD of minicollagen-1 was purified after oxidation according to purification procedure 2. The determination of the NMR structure (PDB accession number 1SP7) was carried out in 5 mm sodium phosphate buffer, pH 6.5, at 15 °C by using homonuclear and heteronuclear techniques and information from weak alignment (44Meier S. Häussinger D. Pokidysheva E. Bächinger H.P. Grzesiek S. FEBS Lett. 2004; (in press)PubMed Google Scholar). Structure representations were generated with MOLMOL (43Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14 (51–55): 29-32Crossref Scopus (6450) Google Scholar). Analytical Ultracentrifugation—Sedimentation equilibrium experiments were performed on a Beckman Optima XL-A analytical ultracentrifuge (Beckman Instruments) equipped with 12-mm Epon double-sector cells in an An-60 Ti rotor. The MCRD was analyzed in 5 mm Tris buffer, pH 7, either without salt or with 100 mm NaCl at 20 °C. The peptide concentrations were adjusted to 0.2–0.8 mm. Sedimentation equilibrium scans were carried out at 48,000 rpm. Molecular masses were evaluated from ln A versus r2 plots, where A is the absorbance at 277 nm and r is the distance from the rotor center. A partial specific volume of 0.73 ml/g was used for all calculations. The MCRD Constitutes a Conserved Sequence Module in Nematocyst Minicollagens and in NOWA—The sequence of minicollagen-1 with domain indications and the alignment of cysteine-rich domains from different minicollagens and NOWA are represented in Fig. 1. Minicollagen-1 consists of an N-terminal MCRD, N-terminal polyproline region, a central collagen sequence, and a shorter C-terminal polyproline stretch followed by a C-terminal MCRD (Fig. 1A). The preceding propeptide is cleaved off during the recombinant expression of minicollagen-1 and probably also in Hydra (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar). As already mentioned in the Introduction the overall sequence homology of the Cys-rich domains is not very high, but the cysteine pattern is identical for all minicollagens and for the eight C-terminal domains of NOWA with the only variation being in the number of residues spacing the first two Cys residues. The sequence of the C-terminal minicollagen-1 MCRD, which has been investigated in the present study, is underlined and the numbering of residues corresponds to the synthetic peptides used in this study (Fig. 1B). Beside the cysteines there is only one conserved residue, which is Pro12 (Fig. 1B, shown in purple). Peptide Synthesis and Oxidation—As the MCRD occurs in different molecular contexts, at the N- and C-terminal extensions of minicollagen molecules as well as eight times repeated at the C terminus of NOWA, we speculated that it might constitute an isolated domain with the capacity of independent folding. Peptide synthesis was carried out for the C-terminal MCRD of minicollagen-1 starting with the last proline residue of the C-terminal polyproline stretch and including the charged C terminus of the full-length molecule. The formation of disulfide bonds occurs in the presence of a redox buffer at 100 μm peptide concentrations to avoid aggregation by intermolecular disulfide bonds (see “Experimental Procedures”). The final product showed a single peak in mass spectroscopic analysis with the reduced and oxidized MCRDs having a difference in molecular weight of 6 Da, thereby strongly indicating the formation of three intramolecular disulfide bonds (Table I). Disulfide bonds can be shown to be all intramolecular in mass spectroscopic analysis. Analytical ultracentrifugation confirmed the absence of significant aggregation or multimerization in solutions from 0.2 to 0.8 mm total peptide concentration (Table I), which was further supported by 1HN NMR T2 relaxation times of more than 100 ms at 25 °C indicative of the prevalence of a monomeric state in solutions of 1.6 mm MCRD.Table IMolar masses of the C-terminal MCRD peptide of minicollagen-1Molar mass UCMolar mass MSMolar mass calculatedg/molg/molg/molMCol1hC reduced25912592MCol1hC oxidized2700 ± 300aAverage of five determinations in the concentration range of 0.2-0.8 mm; S.D. is indicated.25852586a Average of five determinations in the concentration range of 0.2-0.8 mm; S.D. is indicated. Open table in a new tab The NMR Structure of the MCRD—The solution structure of the MCRD was determined independently from two separate peptide preparations (see “Experimental Procedures”). Both structures show an identical tightly packed globular fold (Figs. 2 and 3), which consists of a short N-terminal α-helix between Val5 and Gln9 followed by an inverse γ-turn (Gln9-Val11), a type I β-turn (Val11-Cys14), and a type III β-turn (Pro15-Cys18). Thus cysteines 6, 10, 14, and 18 are directly located in turns, whereas cysteine 2 is located in a proline-rich N-terminal sequence, and cysteine 19 is oriented presumably by the β-III turn and a C-terminal proline. All proline residues in the MCRD are in trans conformation as evidenced by specific nuclear Overhauser effects.Fig. 3Stereo view of the same conformer as in Fig. 2 in ribbon presentation. Only structure 2 is shown. Cysteines and disulfide bonds are shown in yellow and Pro12 is in aquamarine. Disulfide bonds (Cys2-Cys18, Cys6-Cys14 and Cys10-Cys19) as well as the conserved Pro12 are indicated in ball-and-stick representation. Dashed lines indicate the hydrogen bonds identified by MOLMOL. These are bonds between Val5 (O) and Gln9 (HN), Val11 (O) and Cys14 (HN), and Pro15 (O) and Cys18 (HN). The first H-bond belongs to the α-helix, others belong to the three different turns. Pro12 induces a β I turn from residues 11 to 14. All proline residues in the structure can be shown to be in trans conformation from experimental nuclear Overhauser effect distance information.View Large Image Figure ViewerDownload (PPT) The only conserved residue Pro12 (Fig. 1B) imposes a β-I turn topology on residues 11–14 because of its fixed ϕ angle of –60°. Hydrogen bonds are established in the turns between Val5 (O) and Gln9 (HN), Gln9 (O) and Val11 (HN), Val11 (O) and Cys14 (HN), and Pro15 (O) and Cys18 (HN) (Fig. 3), respectively, as derived both from the calculated structures and from the slow 1H/2H exchange upon lyophilization of MCRD and redissolving in 2H2O. The positively charged C terminus of Lys22, Arg23, and Lys24 is flexibly disordered and does not contribute to the MCRD structure (Fig. 2). The first disulfide bond, Cys2-Cys18, clasps the N and C termini of the domain, whereas the Cys6-Cys14 bond connects the N-terminal α-helix to the type I β-turn starting with Val11, thus forming the core of the MCRD structure. The third disulfide bond Cys10-Cys19 is more exposed to the C-terminal surface of the domain and constrains the polypeptide backbone into two consecutive turns. The more surface-exposed N- and C-terminal disulfide bridges represent the most likely candidates for intermolecular disulfide exchange reactions. Complete reduction of the disulfide bonds with an excess of Tris(2-carboxyethyl)phosphine hydrochloride at pH 7.5 results in a complete unfolding without retaining local conformational preferences as well assessed by 1H NMR. Reoxidation of reduced MCRD with an excess of oxidized glutathione at pH 7.5 proceeds extremely fast and is completed within 2 h as observed by 1H NMR spectroscopy (Fig. 4). This is presumably because of the small domain size and the effect of Pro12 upon disulfide bridge formation (see Discussion). Fluorescence spectroscopy allows the monitoring of the refolding due to the quenching of Tyr17 fluorescence by a nearby disulfide bridge and yields a folding half-time of 1.5 h at pH 8 upon oxygen saturation of the solution in agreement with NMR data (not shown). Model of Trimeric Minicollagen-1—It has already been mentioned that minicollagen-1 at native conditions appears to be a non-covalent trimer. A schematic representation of the trimeric minicollagen molecule including the connections of the N- and C-terminal MCRDs to the polyproline type II helices is shown in Fig. 5. A model of trimeric minicollagen-1 was proposed earlier (9Engel U. Pertz O. Fauser C. Engel J. David C.N. Holstein T.W. EMBO J. 2001; 20: 3063-3073Crossref PubMed Scopus (51) Google Scholar) in which the MCRD was assumed to have a linear structure. After elucidation of the structure of the MCRD we are now able to draw a schematic model in scale with the known dimensions of the collagen triple helix and the polyproline-II helix (4Bateman J.F. Lamande S.R. Ramshaw J.A.M. Comper W.D. Collagen Superfamily. Extracellular Matrix. Molecular Components and Interactions. 2. Harwood Academic Publisher, Amsterdam1996Google Scholar). An open question is the geometry of the connections between the collagen and polyproline parts of the molecule and between MCRDs and polyprolines, respectively. The angles and flexibility at the junction sequence APLP in the N terminus and the single Ala spacer in the C terminus (Fig. 1, shown in black) or by any Pro residue that potentially can occur in cis conformation are not known. The lack of association between MCRDs even at relatively high concentrations would suggest that they also do not associate in minicollagen without the help of an isomerase or another catalytic system. Polyproline or polyhydroxyproline-II helices are also known to be monomeric. For these reasons the polyproline arms with their MCRD heads are displayed as non-interacting entities in Fig. 5. This is confirmed by previous biochemical data showing that minicollagen-1 trimers are not disulfide-cross-linked and the triple helix is stabilized solely by the collagen sequence composition (10Özbek S. Pertz O. Schwager M. Lustig A. Holstein T. Engel J. J. Biol. Chem. 2002; 277: 49200-49204Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The ways that MCRDs are linked to the main molecule are shown schematically (Fig. 5, red arrows) in the upper (N-terminal) and the lower (C-terminal) part of Fig. 5. The polyproline-II helices approach the MCRDs from the N terminus for the C-terminal domain and from C terminus for the N-terminal domain. The connections between the polyproline-II helices and the MCRDs might alter the accessibility of particular disulfide bridges in the MCRD. As can be seen from the comparison of Figs. 3 and 5, in the N-terminal MCRD the Cys10-Cys19 disulfide is less exposed. An opposite situation is observed in the C terminus where the polyproline-II stretch hides the Cys2-Cys18 bridge. Accordingly the suggested candidates for intermolecular disulfide exchange are Cys2-Cys18 for the N-terminal and Cys10-Cys19 for the C-terminal MCRD. Small Cys-rich domains are widely distributed building blocks of extracellular proteins in essentially all phyla including plants and bacteria. The most abundant domain type is the epidermal growth factor domain (EGF, smart00181). Including its variants EGFca (smart00179) and EGF-like (smart0001) several thousands of different EGF-domains are known in proteins of different functions. In most cases EGF domains are arranged in arrays with other domains. Laminin (20Engel J. Biochemistry. 1992; 31: 10643-10651Crossref PubMed Scopus (187) Google Scholar) and fibrillin (21Handford P.A. Biochim. Biophys. Acta. 2000; 1498: 84-90Crossref PubMed Scopus (70) Google Scholar) are two of very many examples. Many Cys-rich domains, however, also exist as single autonomous proteins, and the epidermal growth factor domain is a well known example. This fact provides a bridge to the numerous low molar mass Cys-rich proteins, which are also products of larger precursor forms but express their function as toxins, antimicrobial peptides, or other small bioactive agents. Variations of sequences, cysteine patterns, and three-dimensional structures are, however, very large for this diverse class of small proteins, and clear" @default.
• W2103230402 created "2016-06-24" @default.
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• W2103230402 date "2004-07-01" @default.
• W2103230402 modified "2023-10-03" @default.
• W2103230402 title "The Structure of the Cys-rich Terminal Domain of Hydra Minicollagen, Which Is Involved in Disulfide Networks of the Nematocyst Wall" @default.
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