NMR Shows Hydrophobic Interactions Replace Glycine Packing in the Triple Helix at a Natural Break in the (Gly-X-Y) Repeat

Abstract

Little is known about the structural consequences of the more than 20 breaks in the (Gly-X-Y)n repeating sequence found in the long triple helix domain of basement membrane type IV collagen. NMR triple resonance studies of doubly labeled residues within a set of collagen model peptides provide distance and dihedral angle restraints that allow determination of model structures of both a standard triple helix and of a triple helix with a break in solution. Although the standard triple helix cannot continue when Gly is not every third residue, the NMR data support rod-like molecules that have standard triple-helical structures on both sides of a well defined and highly localized perturbation. The GAAVM break region may be described as a “pseudo triple helix,” because it preserves the standard one-residue stagger of the triple helix but introduces hydrophobic interactions at the position normally occupied by the much smaller and hydrogen-bonded Gly residue of the repeating (Gly-X-Y)n sequence. This structure provides a rationale for the consensus presence of hydrophobic residues in breaks of similar length and defines a novel variant of a triple helix that could be involved in recognition. Little is known about the structural consequences of the more than 20 breaks in the (Gly-X-Y)n repeating sequence found in the long triple helix domain of basement membrane type IV collagen. NMR triple resonance studies of doubly labeled residues within a set of collagen model peptides provide distance and dihedral angle restraints that allow determination of model structures of both a standard triple helix and of a triple helix with a break in solution. Although the standard triple helix cannot continue when Gly is not every third residue, the NMR data support rod-like molecules that have standard triple-helical structures on both sides of a well defined and highly localized perturbation. The GAAVM break region may be described as a “pseudo triple helix,” because it preserves the standard one-residue stagger of the triple helix but introduces hydrophobic interactions at the position normally occupied by the much smaller and hydrogen-bonded Gly residue of the repeating (Gly-X-Y)n sequence. This structure provides a rationale for the consensus presence of hydrophobic residues in breaks of similar length and defines a novel variant of a triple helix that could be involved in recognition. Although the collagen triple helix can be considered as one of the most well defined protein motifs, there are still surprises and undefined molecular features. All non-fibrillar collagens contain sites where the repeating Gly-X-Y pattern is interrupted, and evidence suggests that these breaks are of functional importance, playing a role in molecular or higher order structure, or serving as recognition sites for interactions (1Kielty C.M. Grant M.E. Connective Tissue and Its Heritable Disorders, Molecular, Genetic and Medical Aspects, 2nd Ed. 2002; (, pp. , Wiley Liss, New York): 159-222Google Scholar, 2Miles A.J. Knutson J.R. Skubitz A.P. Furcht L.T. McCarthy J.B. Fields G.B. J. Biol. Chem. 1995; 270: 29047-29050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The structural consequences of a break for the triple helix are not understood. The unique triple helix protein motif found in collagen has three extended polyproline II-like chains supercoiled about a common axis. The typical triple helix is characterized by a (Gly-X-Y)n repeating sequence. The three chains are staggered by one residue, and Gly residues must be present as every third residue in each chain so that the three chains can pack very tightly, burying the Gly residues and forming Gly N-H...C=O (X) hydrogen bonds (3Rich A. Crick F.H. J. Mol. Biol. 1961; 3: 483-506Crossref PubMed Scopus (601) Google Scholar, 4Ramachandran G.N. Treatise on Collagen. 1967; (Ramachandran, G. N., ed) pp. , Academic Press, New York: 103-184Google Scholar, 5Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (890) Google Scholar). The abundant fibrillar collagens, e.g. types I, II, and III, maintain this precise Gly-X-Y repeat throughout their ∼1000-residue triple helix domain, and the replacement of even one Gly by another residue results in a pathological condition (1Kielty C.M. Grant M.E. Connective Tissue and Its Heritable Disorders, Molecular, Genetic and Medical Aspects, 2nd Ed. 2002; (, pp. , Wiley Liss, New York): 159-222Google Scholar). But many non-fibrillar collagens have been identified, and these all contain one or more breaks in the repeating Gly-X-Y pattern (1Kielty C.M. Grant M.E. Connective Tissue and Its Heritable Disorders, Molecular, Genetic and Medical Aspects, 2nd Ed. 2002; (, pp. , Wiley Liss, New York): 159-222Google Scholar). The non-fibrillar type IV collagen is found in basement membranes of all multicellular animals, and its network architecture contributes to the essential function of basement membranes in providing mechanical support to cells, serving as semi-permeable barriers between tissues, and providing signals for differentiation (6Schittny J.C. Yurchenco P.D. Curr. Opin. Cell Biol. 1989; 1: 983-988Crossref PubMed Scopus (69) Google Scholar, 7Hudson B.G. Reeders S.T. Tryggvason K. J. Biol. Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar, 8Hudson B.G. Tryggvason K. Sundaramoorthy M. Neilson E.G. N. Engl. J. Med. 2003; 348: 2543-2556Crossref PubMed Scopus (764) Google Scholar, 9Yurchenco P.D. Ruben G.C. Am. J. Pathol. 1988; 132: 278-291PubMed Google Scholar). In type IV collagen, there are more than 20 breaks in the ∼1350-residue-long triple helix of the form Gly-X-Y-(AA)n-Gly-X-Y, and the lengths of the breaks range from common short breaks where n = 1, to long breaks where n = 26 (10Zhou J. Hertz J.M. Leinonen A. Tryggvason K. J. Biol. Chem. 1992; 267: 12475-12481Abstract Full Text PDF PubMed Google Scholar, 11Brazel D. Oberbaumer I. Dieringer H. Babel W. Glanville R.W. Deutzmann R. Kuhn K. Eur. J. Biochem. 1987; 168: 529-536Crossref PubMed Scopus (104) Google Scholar). To begin to define the effects that small breaks have on triple helix structure, NMR studies are presented here on a model peptide of a type IV collagen break where n = 4. Peptide models can provide an approach to characterizing the effects of breaks on triple helix structure and stability (12Baum J. Brodsky B. Curr. Opin. Struct. Biol. 1999; 9: 122-128Crossref PubMed Scopus (120) Google Scholar, 13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Our laboratories recently reported that a 30-mer homotrimer peptide model for a natural break found in the α5(IV) chain of type IV collagen, GPOGAAVMGPOGPO (residues 386–399, where O is used as a single letter code for hydroxyproline (Hyp) 2The abbreviations used are: Hyp, hydroxyproline (three-letter code); O, hydroxyproline (single-letter code); HSQC, heteronuclear single quantum coherence; GFT, G-matrix Fourier Transform; TOCSY, total correlation spectroscopy; PPII, polyproline II; NOE, nuclear Overhauser effect. ) forms a stable trimeric structure, with a relatively small decrease in stability compared with the triple helical structure of a homologous peptide with Gly as every third residue, GPOGAAGVMGPO (13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The classic triple helix structure cannot continue when Gly is not present as every third residue, and biophysical studies indicate that the break decreases the triple helix content, destabilizes the triple helix by 10 °C, and reduces the enthalpy substantially. Although NMR experiments reveal a non-random conformation within the break, it was not clear whether there is a way to continue a modified triple helix through this GAAVM region, or whether a new local structure, such as a β-bend, is introduced. Here NMR triple resonance experiments are presented on a set of doubly labeled peptides with and without the GAAVM break (Table 1), allowing the assignment and tracking of individual chains, the identification of intra- and intermolecular NOEs, and the determination of ϕ dihedral angles through 3JHNHa coupling constants. These NMR parameters are used as restraints in energy minimization of molecular models derived from high resolution x-ray structures of several homologous peptides, to allow the first visualization of the solution molecular conformation of a standard triple helix and of a triple helix with a naturally occurring type IV collagen break. The break in the type IV collagen sequence leads to a highly localized distortion of the triple helix. The GAAVM region has a well defined conformation, which maintains the one-residue stagger but introduces hydrophobic packing of three Val residues at the center of the molecule, which would be normally occupied by Gly residues.TABLE 1Peptide sequences of GAAGVM and GAAVMa15N- and 13C-labeled residues are underlined and bolded Open table in a new tab a15N- and 13C-labeled residues are underlined and bolded Sample Preparation—Peptides Ac-(GPO)4GAAGVM(GPO)4GY-CONH2, designated as the GAAGVM peptide, and Ac-(GPO)4GAAVM(GPO)4GY-CONH2, designated as the GAAVM peptide, were synthesized by Tufts University Core Facility (Boston, MA), as previously described (13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The peptides were made with selectively 13C/15N doubly labeled residues (underlined below): residues Gly13, Ala14, Ala15, Gly16, Val17, and Gly25 were labeled in peptide GAAGVM; residues Gly13, Ala14, Ala15, Val16, and Gly24 were labeled in the peptide GAAVM. Peptides were purified using a Waters XTerra Prep C18 column on an Amersham Biosciences fast-protein liquid chromatography system, and the identity of the peptides was confirmed by for both peptides were in at with of for GAAGVM and for GAAVM. NMR experiments were on a with a and the G-matrix Fourier experiments J. Am. Chem. 2003; PubMed Scopus Google Scholar, J. PubMed Scopus Google were on a with a at the New to and heteronuclear single quantum and a of triple resonance experiments were at °C, M. A. PubMed Scopus Google G. J. 1992; Scholar, W. J. Am. Chem. 1994; Scopus Google M. A. PubMed Scopus Google M. A. J. M. J. B. 1993; Scopus Google A. J. Am. Chem. 1992; Scopus Google and J. Am. Chem. 2003; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). J. 1988; Scholar, G. G. K. J. 1989; with a of and J. 1988; Scholar, G. G. K. J. 1989; Scholar, D. A. J. Am. Chem. 1989; Scopus Google with of were at from in were used to and the data at were used to experiments A. J. Am. Chem. 1993; Scopus Google were to 3JHNHa coupling at °C, with an coupling of The for the 3JHNHa coupling was by A. J. 1992; of on a GAAVM at All data were using the G. J. A. J. 1995; PubMed Scopus Google and with or J. 1994; PubMed Scopus Google Scholar). In the for all a was to the data to with a The data were to and Fourier The and in all experiments were by G. A. J. 1992; by a to and Fourier The data for each In the 3JHNHa the to were to the coupling A. J. Am. Chem. 1993; Scopus Google Scholar). was on the in The was as the standard of the in the of The and the of the 3JHNHa coupling was on the and coupling from the and of with the A. J. 1992; an of was found for the of the GAAVM 1, J. NMR and (, pp. , Academic Press, was to of and the of was The coupling of doubly labeled GAAGVM and GAAVM peptides were by the coupling by where is is and is the coupling of and model structure of GAAGVM was on the structure of J. Brodsky B. Berman H.M. Struct. Biol. 1999; PubMed Scopus Google and using the The residues in were with the residues The structure was with a standard and from residue to residue using the Chem. 2003; PubMed Scopus Google to a of This structure was used to the of the of a standard triple helix were to the model structure of GAAGVM using the J.M. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). The was by to or smaller than and these as and chains and for peptides GAAGVM and GAAVM were made from in the and as and chains and model of GAAGVM was by the ϕ angle restraints from the 3JHNHa with to the energy of 3JHNHa and of an were used to the model with the model of GAAVM was on the x-ray structure of J. J. R. Brodsky B. Berman H.M. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). were to The model was with 3JHNHa coupling restraints to the GAAGVM peptide minimization is very to the structure and to the of the a of structures with were of 3JHNHa and that most structures were not with all of the structure that was with 3JHNHa and all was for GAAGVM and GAAVM. NMR and between GAAVM and GAAGVM both peptides, all could be to chains of the triple helix as in the heteronuclear single quantum by the The assignment is derived from the triple resonance and the chain stagger is derived from experiments that define To the a of triple resonance experiments were on the GAAGVM peptide, where each residue in the of the peptide is labeled (Table the GAAGVM peptide, triple resonance M. A. PubMed Scopus Google G. J. 1992; Scholar, W. J. Am. Chem. 1994; Scopus Google M. A. PubMed Scopus Google and M. J. B. 1993; Scopus Google A. J. Am. Chem. 1992; Scopus Google experiments were (Table were for residues and The from the residue were highly in the and which is to be a of the in of the residues in the sequence. To the three an J. Am. Chem. 2003; PubMed Scopus Google Scholar, J. PubMed Scopus Google was used and the of the of residue and the of residue for the GAAVM peptide were for the doubly labeled in the GAAVM peptide from a single of M. A. J. experiments (Table because there was in the or were confirmed by the M. A. PubMed Scopus Google (Table of peptide GAAGVM and Peptide experiments and that can be from the are with Peptide In experiments and that can be from the are with Open table in a new tab experiments were used to which to the identification of the three chains in of their between chain and chain in GAAGVM and between and in GAAVM the between chains and identification of the 1), chains and chains of the of set the for using distance and dihedral angle in structure The of the GAAGVM peptide a typical pattern for the triple helix. labeled residue at one and one or more with the presence of the triple helix Gly25 a single resonance to the (13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google residues Gly13, Ala14, Ala15, Gly16, and all have for and three for all the to the sequence (13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, Brodsky B. Baum J. 1993; PubMed Scopus Google Scholar, Brodsky B. Baum J. PubMed Scopus Google Scholar). and have similar to each which may their similar in the sequence. The of the GAAVM peptide three as well as for each labeled This that a well defined structure with three chains is present in the region where the Gly-X-Y pattern is within the repeating region at the a single resonance at the typical triple helix position similar to Gly25 in three are for residues Gly13, Ala14, Ala15, and of the of the in for most in GAAVM compared with GAAGVM that there is a in the local of the the Val in the GAAVM peptide have very in both the and with the most from to The may a in the hydrogen of Val in GAAVM to of in a from the GAAGVM in a classic triple helical conformation is from a GAAGVM model The high resolution structures of a of peptides have been by x-ray J. Brodsky B. Berman H.M. Struct. Biol. 1999; PubMed Scopus Google Scholar, J. R. Brodsky B. A. Berman H.M. J. Mol. Biol. PubMed Scopus Google Scholar, K. J. Biochem. 1999; PubMed Scopus Google Scholar, J. Brodsky B. Berman H.M. J. Mol. Biol. PubMed Scopus Google and these structures have confirmed and molecular for the triple-helical model structure of the GAAGVM peptide was by molecular using the structure of the peptide J. Brodsky B. Berman H.M. Struct. Biol. 1999; PubMed Scopus Google because it a sequence with by The residues were with the residues and the structure was for The of this GAAGVM model structure the of the Gly residues are the center of the triple helix and form hydrogen bonds with the of the residue of a chain The of the residues the the of the residues more a chain within the a hydrogen with the of the Gly of the is from the model structure for the GAAGVM peptide and of that are within for the to and in this structure indicate that there is one or more within and indicate the individual of can be from the and in are not The of residue by where to the chain and to the have a of to the and chain residues of than to the and chain residues of are by which are on the short the chain in can be for all residues from to or to are for the to These short from the one residue stagger of the triple helix. are between Gly where the Gly residues in the three chains are in the center in a staggered This staggered packing results in to and and to and unique between Gly to and and between Gly to and are and would to between to and to in GAAVM and was for the data of the GAAGVM peptide, using to the and of and intermolecular to from the GAAGVM model structure The and with the packing of the Gly and the stagger between chains are The chain stagger of and was confirmed from and is a high of in the GAAGVM peptide from the for and in the and as a there are that are not in the experiments when compared with the to the of the and resonance of chains and of Gly16, the were not the it is to the triple helix stagger from the and to that the solution conformation of the GAAGVM peptide is with a typical triple helix similar to the model was for the data for GAAVM to the solution conformation of the peptide with the break to the intra- and intermolecular in the GAAGVM which are as are from The of as were on the of the from the GAAGVM model and a stagger of the triple helix throughout the GAAVM break even a standard triple helix is not to the of the Gly The between the three chains of the residues to to to and to are as well as from to and to the one residue In new between the three chains of the residues at the as well as between the residues and the of residues and are in the GAAVM These new for at the break are similar to the for in the Gly-X-Y repeating sequence of the GAAGVM This suggests that the of is packing the center of the triple helix in this break region, to the of the much smaller Gly residue found at the position as the Val in the GAAGVM of NMR were to 3JHNHa coupling for both the GAAGVM and GAAVM peptides to the range of dihedral angles that can be in the break with in a standard triple helix with a Gly-X-Y repeat GAAGVM the 3JHNHa coupling of Ala14, Ala15, and one of the of Gly13, Gly16, and Gly24 are relatively in the range of Val to have a in the range of 3JHNHa of a and determination of a coupling ϕ angles from the A. J. Am. Chem. 1993; Scopus Google 3JHNHa = of the solution of which is with Gly dihedral angles ϕ of for that the ϕ angles for the labeled residues in the GAAGVM peptide are with the ϕ angles found in the polyproline conformation J. Brodsky B. Berman H.M. Struct. Biol. 1999; PubMed Scopus Google Scholar). In the GAAVM peptide not have coupling the peptide of and as well as the of Gly24 at the are very similar to in The coupling of and in the peptide are in the range of from the to in are to the for of that ϕ angles of and The of the of the coupling between GAAGVM and GAAVM indicate that the distortion of the ϕ angles is very local and that and angles the of the GAAGVM and GAAVM ϕ angle restraints from 3JHNHa were used to the model of GAAGVM derived from an x-ray structure as described The model not from the that the model derived from x-ray structure is in with NMR and In the GAAGVM all angles the region of the with a triple helix The between the model from an x-ray structure and the NMR solution data for the GAAGVM peptide a to NMR data to define the conformation in solution for a GAAVM break. this peptide, a model was on a x-ray structure with a break in the Gly-X-Y repeating sequence J. J. R. Brodsky B. Berman H.M. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). Our peptide from the peptide by x-ray in a break compared with a break and in a natural type IV collagen sequence compared with the repeating sequence. The sequence of the structure peptide was by and the structure was ϕ angle restraints from GAAVM NMR 3JHNHa The structure with NMR The of the model preserves the stagger between the chains and introduces packing of Val the and the Val residues are to the center the small Gly The three Val residues form a small localized hydrophobic in GAAVM a of localized in the dihedral angles of residues the three Val can be in this the rod-like structure without a The GAAVM region may be as a helix,” because it the stagger and the packing of the chains and in the dihedral angles of a small of The of one residue in GAAVM GAAGVM a of of the triple helix on both sides of the break, as in the structure for peptides with breaks J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (890) Google Scholar, J. J. R. Brodsky B. Berman H.M. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). The of the GAAVM model a local of the dihedral angles to the model of The dihedral angles are the most with all three chains of the range In the dihedral angles of and to and all are the The highly localized of the is by the that residues in the chains are from standard collagen dihedral that the well defined in structure is by a of residues in the break so that one can the of triple helix structure on both sides of the break. of of support this GAAVM model by molecular with NMR of the 3JHNHa and NMR from the model in and to the 3JHNHa and indicate that the model has a to the data not models not with the the GAAVM model is not it provides a of the structure that can be by the peptide the break that the as described it the consensus sequence of The NMR data on the GAAVM peptide in solution is with the rod-like of the triple and the highly localized of the structural of x-ray structures on peptides with small breaks J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (890) Google Scholar, J. J. R. Brodsky B. Berman H.M. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). The at the break reported for the structure is with the hydrogen at in GAAGVM GAAVM 13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google and data not GAAVM break here one of the most common types of break in type IV collagen and non-fibrillar collagens, with residues in a The consensus pattern for such breaks as a small residue at the position and a hydrophobic residue at the position (13Mohs A. Popiel M. Li Y. Baum J. Brodsky B. J. Biol. Chem. 2006; 281: 17197-17202Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The GAAVM break the consensus with an residue at the position and a Val residue at the The model structure derived from NMR data suggests that the of a hydrophobic residue at the position of the break novel hydrophobic packing the of the the presence of a smaller residue is the hydrophobic residue to the distortion for this hydrophobic The presence of a small hydrophobic by the Val residues at position suggests that hydrophobic can for the Gly packing normally found at the position and this and similar breaks can be present in non-fibrillar collagens with similar to of fibrillar The structural consequences of the GAAVM break as a for a typical break of this in non-fibrillar collagens, which are such as type collagen in and type collagen in (1Kielty C.M. Grant M.E. Connective Tissue and Its Heritable Disorders, Molecular, Genetic and Medical Aspects, 2nd Ed. 2002; (, pp. , Wiley Liss, New York): 159-222Google Scholar). The breaks in these homotrimer collagens are to the triple helix structure reported here for the model non-fibrillar collagens, such as type IV collagen in basement are of or three chain type IV collagen a break in one chain is a break in the chains, a structure similar to that in the model in most the break in one type IV collagen chain is another of break or an sequence in the chains, and it is not clear whether such have or effects on the triple helix for NMR experiments on the GAAVM peptide and for with of triple resonance the New for to their NMR and for in the G-matrix Fourier NMR