Hydroxylation-induced Stabilization of the Collagen Triple Helix

4(R)-Hydroxyproline in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence of collagen plays a crucial role in the stability of the triple helix. Since the peptide (4(R)-Hyp-Pro-Gly)10 does not form a triple helix, it was generally believed that polypeptides with a -Gly-4(R)-Hyp-Yaa-repeated sequence do not form a triple helix. Recently, we found that acetyl-(Gly-4(R)-Hyp-Thr)10-NH2 forms a triple helix in aqueous solutions. To further study the role of 4(R)-hydroxyproline in the Xaa position, we made a series of acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptides where Yaa was alanine, serine, valine, and allo-threonine. We previously hypothesized that the hydroxyl group of threonine might form a hydrogen bond to the hydroxyl group of 4(R)hydroxyproline. In water, only the threonine- and the valine-containing peptides were triple helical. The remaining peptides did not form a triple helix in water. In 1,2- and in 1,3-propanediol at 4 °C, all the soluble peptides were triple helical. From the transition temperature of the triple helices, it was found that among the examined residues, threonine was the most stable residue in the acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptide. The transition temperatures of the valine- and allo-threonine-containing peptides were 10 degrees lower than those of the threonine peptide. Surprisingly, the serine-containing peptide was the least stable. These results indicate that the stability of these peptides depends on the presence of a methyl group as well as the hydroxyl group and that the stereo configuration of the two groups is essential for the stability. In the threonine peptide, we hypothesize that the methyl group shields the interchain hydrogen bond between the glycine and the Xaa residue from water and that the hydroxyl groups of threonine and 4(R)hydroxyproline can form direct or water-mediated hydrogen bonds. 4(R)-Hydroxyproline in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence of collagen plays a crucial role in the stability of the triple helix. Since the peptide (4(R)-Hyp-Pro-Gly)10 does not form a triple helix, it was generally believed that polypeptides with a -Gly-4(R)-Hyp-Yaa-repeated sequence do not form a triple helix. Recently, we found that acetyl-(Gly-4(R)-Hyp-Thr)10-NH2 forms a triple helix in aqueous solutions. To further study the role of 4(R)-hydroxyproline in the Xaa position, we made a series of acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptides where Yaa was alanine, serine, valine, and allo-threonine. We previously hypothesized that the hydroxyl group of threonine might form a hydrogen bond to the hydroxyl group of 4(R)hydroxyproline. In water, only the threonine- and the valine-containing peptides were triple helical. The remaining peptides did not form a triple helix in water. In 1,2- and in 1,3-propanediol at 4 °C, all the soluble peptides were triple helical. From the transition temperature of the triple helices, it was found that among the examined residues, threonine was the most stable residue in the acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptide. The transition temperatures of the valine- and allo-threonine-containing peptides were 10 degrees lower than those of the threonine peptide. Surprisingly, the serine-containing peptide was the least stable. These results indicate that the stability of these peptides depends on the presence of a methyl group as well as the hydroxyl group and that the stereo configuration of the two groups is essential for the stability. In the threonine peptide, we hypothesize that the methyl group shields the interchain hydrogen bond between the glycine and the Xaa residue from water and that the hydroxyl groups of threonine and 4(R)hydroxyproline can form direct or water-mediated hydrogen bonds. Collagen is the most abundant protein in multicellular animals. Collagens work not only as the scaffold of tissues and organs but also as regulators of many biological process including cell attachment, cell proliferation, and gene expression. Although it is one of the most extensively studied proteins, the structure of the collagen triple helix is still not fully understood. Collagen molecules consist of three polyproline II-like left-handed helices (all trans) that form a right-handed super helical structure, the triple helix. Formation of a triple helix requires the presence of a repeated -Gly-Xaa-Yaa-sequence. The Xaa and Yaa positions are frequently occupied by proline residues. Almost all prolines in the Yaa position of vertebrate collagens are posttranslationally modified to 4(R)-hydroxyproline by prolyl-4-hydroxylase (E.C. 1.14.11.2). Although there are some exceptions, the transition temperature of the collagen triple helix from various species is correlated to the 4(R)-Hyp content (1Burjanadze T.V. Biopolymers. 1979; 18: 931-938Crossref PubMed Scopus (111) Google Scholar, 2Burjanadze T.V. Veis A. Connect. Tissue Res. 1997; 36: 347-365Crossref PubMed Scopus (31) Google Scholar). Previous studies have shown that the peptide (Pro-4(R)-Hyp-Gly)10 forms a triple helix and has a significantly higher transition temperature than the trimer of the peptide (Pro-Pro-Gly)10 (3Engel J. Chen H.T. Prockop D.J. Klump H. Biopolymers. 1977; 16: 601-622Crossref PubMed Scopus (208) Google Scholar). Several mechanisms were proposed for the stabilizing effect of 4(R)-hydroxyproline in the Yaa position (4Vitagliano L. Berisio R. Mazzarella L. Zagari A. Biopolymers. 2001; 58: 459-464Crossref PubMed Scopus (196) Google Scholar, 5Vitagliano L. Berisio R. Mastrangelo A. Mazzarella L. Zagari A. Protein Sci. 2001; 10: 2627-2632Crossref PubMed Scopus (133) Google Scholar, 6Kramer R.Z. Bella J. Brodsky B. Berman H.M. J. Mol. Biol. 2001; 311: 131-147Crossref PubMed Scopus (181) Google Scholar, 7Jenkins C.L. Raines R.T. Nat Prod. Rep. 2002; 19: 49-59Crossref PubMed Scopus (208) Google Scholar, 8Gough C.A. Anderson R.W. Bhatnagar R.S. J. Biomol. Struct. Dyn. 1998; 15: 1029-1037Crossref PubMed Scopus (14) Google Scholar, 9Brodsky B. Ramshaw J.A. Matrix Biol. 1997; 15: 545-554Crossref PubMed Scopus (398) Google Scholar). Neither (Pro-4(S)-Hyp-Gly)10, (4(S)-Hyp-Pro-Gly)10, nor (4(R)Hyp-Pro-Gly)10 form a stable triple helix in water (10Inouye K. Sakakibara S. Prockop D.J. Biochim. Biophys. Acta. 1976; 420: 133-141Crossref PubMed Scopus (109) Google Scholar, 11Inouye K. Kobayashi Y. Kyogoku Y. Kishida Y. Sakakibara S. Prockop D.J. Arch. Biochem. Biophys. 1982; 219: 198-203Crossref PubMed Scopus (107) Google Scholar). Since prolyl residues seem the most stable among the natural amino acids in the triple helix, it has been believed that (Gly-4(R)-Hyp-Yaa)10 peptides (Yaa is any amino acid residue) do not form a triple helix in water, until our report showed that the peptide Ac-(Gly-4(R)-Hyp-Thr)10-NH2 forms a triple helix in water (12Bann J.G. Bächinger H.P. J. Biol. Chem. 2000; 275: 24466-24469Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The thermal stability of the collagen triple helix arises from the interchain hydrogen bonds between the amide group of glycine and the carboxyl group of Xaa and the restriction of the ϕ dihedral of the pyrrolidine ring structure. In addition, several additional factors have been reported to contribute to the stability of the triple helix: solvent water molecule-mediated hydrogen bonds (13Bella J. Brodsky B. Berman H.M. Structure. 1995; 3: 893-906Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar), the propensity of the pyrrolidine ring puckering down (Cγ-endo pucker) in the Xaa and up (Cγ-exo pucker) in the Yaa position (5Vitagliano L. Berisio R. Mastrangelo A. Mazzarella L. Zagari A. Protein Sci. 2001; 10: 2627-2632Crossref PubMed Scopus (133) Google Scholar, 14Berisio R. Vitagliano L. Mazzarella L. Zagari A. Biopolymers. 2000; 56: 8-13Crossref PubMed Scopus (100) Google Scholar, 15Berisio R. Vitagliano L. Sorrentino G. Carotenuto L. Piccolo C. Mazzarella L. Zagari A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 55-61Crossref PubMed Scopus (28) Google Scholar, 16Berisio R. Vitagliano L. Mazzarella L. Zagari A. Protein Sci. 2002; 11: 262-270Crossref PubMed Scopus (255) Google Scholar), the inductive effect of the hydroxyl group of 4(R)-Hyp to stabilize the trans X-Hyp conformation and strengthen the hydrogen bond (7Jenkins C.L. Raines R.T. Nat Prod. Rep. 2002; 19: 49-59Crossref PubMed Scopus (208) Google Scholar, 17Holmgren S.K. Taylor K.M. Bretscher L.E. Raines R.T. Nature. 1998; 392: 666-667Crossref PubMed Scopus (426) Google Scholar), and the gauche effect of 4(R)-Hyp in the Yaa to pucker up (7Jenkins C.L. Raines R.T. Nat Prod. Rep. 2002; 19: 49-59Crossref PubMed Scopus (208) Google Scholar). However, it is still not possible to consistently explain all the experimental results. Collagens of invertebrates have been shown to exhibit unusual and interesting properties (18Coyne K.J. Qin X.X. Waite J.H. Science. 1997; 277: 1830-1832Crossref PubMed Scopus (226) Google Scholar, 19Qin X.X. Coyne K.J. Waite J.H. J. Biol. Chem. 1997; 272: 32623-32627Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 20Waite J.H. Qin X.X. Coyne K.J. Matrix Biol. 1998; 17: 93-106Crossref PubMed Scopus (192) Google Scholar). An example of this is the cuticle collagen from the deep-sea hydrothermal vent worm Riftia pachyptila. This organism lives under extreme conditions (high pressure, low oxygen, and steep temperature gradients) but is protected from its environment by a thick cuticle (21Gaill F. Mann K. Wiedemann H. Engel J. Timpl R. J. Mol. Biol. 1995; 246: 284-294Crossref PubMed Scopus (65) Google Scholar, 22Gaill F. Wiedemann H. Mann K. Kühn K. Timpl R. Engel J. J. Mol. Biol. 1991; 221: 209-223Crossref PubMed Scopus (48) Google Scholar). The R. pachyptila cuticle is mainly composed of a collagen that forms a plywood-like network of fibrils and exhibits a unique amino acid composition (22Gaill F. Wiedemann H. Mann K. Kühn K. Timpl R. Engel J. J. Mol. Biol. 1991; 221: 209-223Crossref PubMed Scopus (48) Google Scholar, 23Mann K. Mechling D.E. Bächinger H.P. Eckerskorn C. Gaill F. Timpl R. J. Mol. Biol. 1996; 261: 255-266Crossref PubMed Scopus (60) Google Scholar). Characteristic for these collagens is the occurrence of 4(R)-Hyp in the Xaa position of the -Gly-Xaa-Yaa-tripeptide repeat (24Goldstein A. Adams E. J. Biol. Chem. 1970; 245: 5478-5483Abstract Full Text PDF PubMed Google Scholar, 25Muir L. Lee Y.C. J. Biol. Chem. 1970; 245: 502-509Abstract Full Text PDF PubMed Google Scholar). We recently found that the Ac-(Gly-4(R)-Hyp-Thr)10-NH2 peptide forms a triple helix in water, and the addition of galactosyl residue to threonine increases the stability of the triple helix (12Bann J.G. Bächinger H.P. J. Biol. Chem. 2000; 275: 24466-24469Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 26Bann J.G. Peyton D.H. Bächinger H.P. FEBS Lett. 2000; 473: 237-240Crossref PubMed Scopus (71) Google Scholar). To further characterize the mechanism of stabilization of peptides with 4(R)Hyp in the Xaa position, we made a series of polypeptides with the amino acid sequences of acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 (Yaa = Ser, Val, Ala, alloThr) 1The abbreviations used are: alloThr, (2S,3S)-2-amino-3-hydroxybutyric acid, l-allo-threonine; Fmoc, N-(9-fluoroenyl)methoxycarbonyl; tBu, t-butyl. and characterized them by circular dichroism measurements in water, in 1,2- and 1,3-propanediol. The experimental results indicate that the stereo chemical configuration of both hydroxyl and methyl groups of threonine are essential in stabilizing the triple helix in these peptides. Peptide Synthesis and Purification—Peptides were synthesized either on a Milligen 9050 peptide synthesizer or on an ABI 433A (Applied Biosystems, Foster City, CA). Couplings were carried out on PALPEG-PS resin (PerSeptive Biosystems, 0.16 mmol/g) using Fmoc amino acids (Fmoc-Gly-OH, Fmoc-4(R)-Hyp(tBu)-OH, Fmoc-Thr-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-4(R)Hyp (Novabiochem), and acetyl glycine (Bachem, Torrence, CA). Fmoc-Gly-4(R)Hyp-OH was also synthesized from H-Gly-4(R)-Hyp-OH (Bachem) and (N-(9-fluorenylmethyloxycarbonyloxy)-succinimide) (Bachem). O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (Perseptive Biosystems) (4.0 eq)/diisopropylethylamine-mediated peptide couplings. The peptides were cleaved from the resin and purified by semipreparative high performance liquid chromatography (Vydac® C18, 5 μm, 300E, 250 × 10 mm, W.R. Grace, Columbia, MD). All synthesized peptides were characterized by amino acid analysis and matrix-assisted laser desorption/ionization/time-of-flight mass spectrometry. Circular Dichroism Spectroscopy—Circular dichroism spectra were recorded on an Aviv 202 spectropolarimeter using a Peltier thermostatted cell holder and a 1-mm (Starna Cells Inc., Atascadero, CA) or a 0.1-mm (Hellma, Müllheim, Germany) path length rectangular cell. Measurements were performed in water, 1,2-propanediol (Sigma), or 1,3-propanediol (Sigma). Molecular sieve type 3 Å 4–8 mesh beads (EM science) were added to both 1,2- and 1,3-propanediol to remove water before use. Peptide concentrations were determined by amino acid analysis. The wavelength spectra represent at least an average of 10 scans with 0.1-nm wavelength steps. Thermal transitions were recorded at a heating rate of 10 °C/h. Differential Scanning Calorimetry—The temperature dependence of the partial heat capacity was measured in an N-DSC II differential scanning calorimeter (Calorimetry Science Corp., UT). The peptide solutions in 1,2-propanediol were centrifuged at 4 °C for 60 min at 100,000 × g in a Beckman model L-8 ultracentrifuge before measurement. The heating rate was 15 °C/h, and the data were collected and analyzed using the software provided by the manufacturer. Molecular Modeling—The structure was modeled using the structure of Protein Data Bank accession number 1G9W as a template for the backbone conformation (4Vitagliano L. Berisio R. Mazzarella L. Zagari A. Biopolymers. 2001; 58: 459-464Crossref PubMed Scopus (196) Google Scholar, 15Berisio R. Vitagliano L. Sorrentino G. Carotenuto L. Piccolo C. Mazzarella L. Zagari A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 55-61Crossref PubMed Scopus (28) Google Scholar). The carboxyl terminus of (Pro-Pro-Gly)10 was changed to an amide, and the Xaa and Yaa positions were replaced by 4(R)-hydroxyproline and threonine. Side chain conformations of the threonine residues were optimized by manually selecting the lowest energy conformer with the Biopolymer program in Insight II (Accelrys, Pharmacopeia Inc., Princeton, NJ). Subsequent energy minimizations were performed with the Discover program of Insight II, using the consistent valence force field. The backbone was tethered in place throughout with a force constant of 100 kcal/Å2. The peptides were soaked with a five-molecule-thick layer of water. The side chains of all amino acids were uncharged to approximate the effects of solvent shielding (27Vitagliano L. Nemethy G. Zagari A. Scheraga H.A. Biochemistry. 1993; 32: 7354-7359Crossref PubMed Scopus (54) Google Scholar). The protocol for minimization was as follows: the method of steepest descents was used until a maximum derivative of <5 kcal/Å was reached, with the charge term included. Next, the method of conjugate gradients was used for 500 iterations until a maximum derivative of <5.0 kcal/Å was reached, with the charge term included. Next, the method of conjugate gradients was used until a maximum derivative of kcal/Å was reached, with and included. the method was used until a maximum derivative of kcal/Å was with and a bond included. The spectra of collagen molecules a of degrees and a maximum of degrees the presence of a left-handed all trans polyproline II structure. peptides are to spectra form a triple helix. the at that of the peptides measured in water, only the threonine and the peptides showed a at The serine, the alanine, and the peptides showed at the temperature dependence of the at was only the threonine and the peptides showed the transition from triple helix to The peptides showed a of the with temperature was that the peptide than one at 4 °C the of the peptide to a transition The of triple helix of the peptide that the presence of a hydroxyl group is not to stabilize a triple helix with 4(R)-Hyp in the Xaa position in water. Although the methyl group to contribute to the stability in water, it also does not for the stability found in the threonine peptide. groups are to form a stable triple helix. The of triple helix of the peptide also that the configuration of the two groups of threonine significantly to the stability of the peptide. To the thermal stability of all the we to a solvent where a stable triple helix for all peptides. 1,2-propanediol was shown to the temperature of we used this solvent to the thermal stability of these peptides (3Engel J. Chen H.T. Prockop D.J. Klump H. Biopolymers. 1977; 16: 601-622Crossref PubMed Scopus (208) Google Scholar). Previous studies have shown that with than one hydroxyl as and the stability for type collagen at K. S. J. Biochem. PubMed Scopus Google Scholar). results were for with Ac-(Gly-4(R)-Hyp-Thr)10-NH2 and peptides J.G. Peyton D.H. Bächinger H.P. FEBS Lett. 2000; 473: 237-240Crossref PubMed Scopus (71) Google Scholar), 1,2-propanediol for (Pro-Pro-Gly)10 and (Pro-4(R)-Hyp-Gly)10 (3Engel J. Chen H.T. Prockop D.J. Klump H. Biopolymers. 1977; 16: 601-622Crossref PubMed Scopus (208) Google Scholar), and for and or peptides Y. G. Biochemistry. 1997; 36: PubMed Scopus (54) Google Scholar). In the peptides that do not form triple helix in water a triple helix, with the of the peptide that was not soluble in the spectra of peptides in All the soluble peptides and also showed a transition the temperature was In the threonine peptide is still most stable. The of transition temperatures in 1,2-propanediol is = = = = We also measured that does not form a triple helix in water J.G. Peyton D.H. Bächinger H.P. FEBS Lett. 2000; 473: 237-240Crossref PubMed Scopus (71) Google Scholar). The of the thermal transition is at This is a higher than the of °C determined for K. Mechling D.E. Bächinger H.P. Eckerskorn C. Gaill F. Timpl R. J. Mol. Biol. 1996; 261: 255-266Crossref PubMed Scopus (60) Google Scholar). These data indicate that 1,2-propanediol is a stabilizing solvent not only for (Pro-Pro-Gly)10 and but also for the peptides. the data determined from the transition of the peptides in data of the triple helix transition of the peptides in temperatures are and are determined from the measurements with and using in temperatures are determined by differential scanning at the peptide was not soluble at the higher concentrations for differential scanning temperatures are and are determined from the measurements with and using in J. Chen H.T. Prockop D.J. Klump H. Biopolymers. 1977; 16: 601-622Crossref PubMed Scopus (208) Google temperatures are determined by differential scanning at the The peptide was not soluble at the higher concentrations for differential scanning in a the spectra of the peptides in 1,3-propanediol. all peptides showed a and the transition are shown in The peptides are stable in 1,3-propanediol than in The peptide was not soluble in this and was for the peptide. The peptide was the most stable one = by the = the = and the peptide = The structure of the threonine peptide that the methyl group of threonine the of the triple helix between the peptide bond of of the chain and the glycine of the chain The methyl group the interchain hydrogen bond between the group of in the Xaa position of the chain and the amino group of the glycine residue in the The hydroxyl group the of the triple helix. The pyrrolidine ring of in the Xaa position is puckering down the to the threonine The between the of the hydroxyl group of threonine and the of the hydroxyl group of is is than the found in hydrogen bonds We have recently shown that Ac-(Gly-4(R)-Hyp-Thr)10-NH2 forms a stable triple helix in water. This was a it was that 4(R)-hydroxyproline in the Xaa position the of a triple helix it was shown that (4(R)-Hyp-Pro-Gly)10 was to form this structure. the for cuticle it is reported that the sequence for of the content and that the cuticle collagen of R. pachyptila has a low proline and content The stabilizing in the cuticle collagen of R. pachyptila is the of in the Yaa However, the presence of 4(R)-hydroxyproline in the Xaa results with peptides 4(R)-hydroxyproline in the Xaa position and serine, valine, alanine, and some the stabilizing of these peptides. In our studies (12Bann J.G. Bächinger H.P. J. Biol. Chem. 2000; 275: 24466-24469Abstract Full Text Full Text PDF PubMed Scopus (83) Google with the peptide, we that there an additional hydrogen bond in this peptide, as by an in the between the proline and peptides. However, the serine-containing peptide in this study does not form a stable triple helix in water. In we a lower in for the as with the threonine peptide, and this with a the serine-containing peptide significantly stable. The is that the hydroxyl group does not for the stability. The valine-containing peptide is as stable as the peptide in water, that the methyl group plays an role in stabilizing the triple helix as The peptide that the of the hydroxyl and methyl group is in this In water, the peptide does not form a triple helix, and the in 1,2-propanediol is significantly lower than that of the peptide. The effect of both 1,2- and 1,3-propanediol on thermal stability of the is shown in and also has the effect on some of the peptides used in this study not is also to the temperature of type collagen K. S. J. Biochem. PubMed Scopus Google Scholar, E. S. Sci. S. A. 2002; PubMed Scopus Google Scholar). In the temperature of type II collagen is by the addition of H.P. 10: PubMed Scopus Google Scholar). The stabilizing effect of 1,2-propanediol on the (Pro-Pro-Gly)10 and was reported (3Engel J. Chen H.T. Prockop D.J. Klump H. Biopolymers. 1977; 16: 601-622Crossref PubMed Scopus (208) Google Scholar). The mechanism of stabilization by these is still analysis of the in by Berisio R. Vitagliano L. Mazzarella L. Zagari A. Protein Sci. 2002; 11: 262-270Crossref PubMed Scopus (255) Google Scholar), up to that many water molecules with the peptide. These that the high of with the triple helix are the of the is still water or solvent molecules contribute to the stability of the triple helix J. Prockop D.J. Matrix Biol. 1998; 17: PubMed Scopus Google Scholar). However, studies and our experimental results indicate that both 1,2- and 1,3-propanediol significantly contribute to the stability of the triple helix. the peptides are stable in 1,3-propanediol. This is for the peptide, a that is °C higher than in We hypothesize that 1,2-propanediol and 1,3-propanediol molecules might as three or water with the of the triple helix. The in stability in 1,3-propanediol 1,2-propanediol results from the in the of the hydroxyl 1,3-propanediol can form than 1,2-propanediol and can also with the This explain the stability of the peptide in 1,3-propanediol. The effect on the examined peptides that molecules with the chain groups of or the Yaa position residues. The of stability of the threonine peptide, by the valine, and that the configuration of both the methyl and the hydroxyl group is for the stability. The energy minimization that the methyl group of threonine shields the interchain hydrogen bond between the amino group of glycine and the carboxyl group of from solvent This interchain hydrogen bond is a of the thermal stability of the collagen triple helix. of solvent molecules has also been for the peptides J.G. Bächinger H.P. Peyton D.H. Biochemistry. PubMed Scopus Google Scholar). The stability of the threonine peptide as with the peptide the of a by hydrogen bond of the hydroxyl group of threonine. In the structure, the of a direct hydrogen bond between the hydroxyl groups of and threonine a water-mediated hydrogen bond The stability of the peptide that a and that it is We for and for amino acid