Stepwise construction of triple-helical heparin binding sites using peptide models

The specific localization of the asymmetric form of acetylcholinesterase (AChE) in neuromuscular junctions results from the interaction of its collagen-like tail with heparan sulfate proteoglycans in the synaptic basal lamina. This interaction involves two heparin binding consensus sequences of the form XBBXB, where B is a basic residue, located in the triple-helical collagen tail: GRKGR for the N-terminal site and GKRGK for the C-terminal site. To explore the basis of the higher heparin affinity seen for the C-terminal site vs. the N-terminal site, two homologous series of (Gly-Xaa-Yaa)(8) peptides were constructed to model these triple-helical binding sites. Individual tripeptide units from each heparin binding site were introduced in a stepwise fashion into a Gly-Pro-Hyp framework, until the consensus sequence and its surrounding triplets were recreated. As each additional triplet from the binding site is inserted to replace a host Gly-Pro-Hyp triplet, the triple-helix stability decreases, and the drop in thermal stability is close to that expected if each Gly-X-Y triplet contributed independently to global stability. CD spectroscopy and calorimetry show the stability of these AChE model peptides is increased by addition of heparin, confirming binding to heparin, and the lack of significant enthalpy change indicates the binding is largely electrostatic in nature. Displacement assays measure the strength of the peptide-heparin interaction, and indicate an inverse correlation between the peptide ability to bind heparin and its thermal stability. The model peptides for the C-terminal binding site show a greater heparin affinity than the peptide models for the N-terminal binding site only when residues surrounding the consensus sequence are included.     

Triple-helical peptides: an approach to collagen conformation, stability, and self-association

Peptides have been an integral part of the collagen triple-helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence-structure-function relationships. High resolution structures of triple-helical peptides have confirmed the basic Ramachandran triple-helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple-helix stability on the residues in its (Gly-X-Y)(n) repeating sequence has been investigated by measuring melting temperatures of host-guest peptides and an on-line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly-X-Y)(n) amino acid sequence pattern are found in the triple-helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod-like triple-helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple-helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple-helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self-association to the supramolecular structures found in tissues.     

Stability junction at a common mutation site in the collagenous domain of the mannose binding lectin

Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases.     

Nuclear magnetic resonance and circular dichroism studies of a triple-helical peptide with a glycine substitution.

The triple-helical conformation has the stringent amino acid sequence constraint that every third residue must be a glycine, (X-Y-Gly)n. We use nuclear magnetic resonance and circular dichroism to quantify the consequences of a substitution in the glycine position of a triple-helical peptide, and to enhance our understanding of interactions in this basic structural motif. A 30-residue peptide with a Gly----Ala change forms a stable trimer at a folding rate somewhat less than that of the unsubstituted peptide, and the substitution results in a marked decrease in thermal stability and a conformational perturbation of about 30% of the triple-helical structure. Two models were generated for this peptide, one with the alanine residues packed inside the triple helix and one with a looping out of the chain at the substitution site. Studies on the Gly----Ala peptide are useful in understanding connective tissue diseases which result from the substitution of one glycine residue in the triple-helix of fibrillar collagens.     

Design, expression and characterization of collagen-like proteins based on the cell adhesive and crosslinking sequences derived from native collagens

Two recombinant collagen-like proteins consisting of cell adhesion domains derived from native type I collagen were designed and synthesized by a genetic engineering method. The cross-linking sequence, GPPGPCCGGG, derived from collagen III was used to promote triple helix formation through the disulfide bonds formed among three chains by flanking the peptide at the C-terminal of the collagen-like proteins. SDS-PAGE and western-blotting data suggested possibility of the formation of a triple helix structure for both recombinant proteins. CD spectra and thermal stability analyses indicated that the triple-helix structure in the collagen-like proteins was pH-dependent and stabilized under acidic environmental condition. Moreover, the collagen-like protein flanked with the cross-linking sequence at the C-terminal showed the most stable triple-helical conformation under acidic conditions.     

Structural analysis of the heparin-binding site of the NC1 domain of collagen XIV by CD and NMR.

Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an alpha-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic alpha-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial alpha-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG.     

The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13: SEQUENCE SPECIFICITY FOR BINDING AND CLEAVAGE*

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly⁷⁷⁵–Leu⁷⁷⁶ in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

Structural requirements for heparin/heparan sulfate binding to type V collagen

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.     

Structural consequences of D-amino acids in collagen triple-helical peptides

The effects of racemization of aspartic acid on triple-helical formation have been studied using a "host-guest" peptide approach where selected guest Gly-Xaa-Yaa triplets were included within a common acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide frame-work. Four guest triplets, Gly-Asp-Hyp and Gly-Asp-Ala where Asp is either L-Asp or D-Asp were studied. Thermal stability data indicated that incorporation of D-Asp residues prevented triple-helix formation in phosphate buffered saline, although triple-helical structures were formed in a stabilizing solvent, 67% aqueous ethylene glycol. In this solvent the melting temperatures of D-Asp containing peptides were more than 30 degrees C lower than the corresponding peptides containing L-Asp. For Gly-Asp-Ala peptides, but not Gly-Asp-Hyp, peptides, melting profiles indicated that a mixture of the D- and L-Asp containing peptides were able to form heterotrimer triple-helical molecules. These studies illustrate the dramatic destabilizing effect of D-amino acids on the triple-helix stability, but indicate that they can be accommodated in this conformation.     

A statistically derived parameterization for the collagen triple-helix

The triple-helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo- or hetero-tripeptide with a repeating primary sequence of (Gly-X-Y)(n), displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple-helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple-helical; however, no high-resolution structure of any entire protein is yet available. Given the drastic morphological differences in self-assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple-helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high-resolution X-ray crystal structures of model triple-helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and C(beta) atom locations for the triple-helix. Unlike existing homology models, this allows easy prediction of an entire triple-helix structure based on all existing high-resolution triple-helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.     

Conformational effects of Gly-X-Gly interruptions in the collagen triple helix

The collagen model peptide with sequence (Pro-Hyp-Gly)4-Pro-Gly-(Pro-Hyp-Gly)5 contains a central Gly-Pro-Gly interruption in the consensus collagen sequence. Its high-resolution crystal structure defines the molecular consequences of such an interruption for the collagen triple-helical conformation, and provides insight into possible structural and biological roles of similar interruptions in the -Gly-X-Y- repeating pattern found in non-fibrillar collagens. The peptide (denoted as the Hyp minus peptide or Hyp-) forms a rod-like triple helix structure without any bend or kink, and crystallizes in a quasi-hexagonal lattice. The two Pro-Hyp-Gly zones adopt the typical triple-helical collagen conformation with standard Rich and Crick II hydrogen bonding topology. Notably, the central zone containing the Gly-Pro-Gly interruption deviates from the standard structure in terms of hydrogen bonding topology, torsion angles, helical, and superhelical parameters. These deviations are highly localized, such that the standard features are regained within one to two residues on either side. Conformational variations and high temperature factors seen for the six chains of the asymmetric unit in the zone around the interruption point to the presence of a local region of considerable plasticity and flexibility embedded within two highly rigid and ordered standard triple-helical segments. The structure suggests a role for Gly-X-Gly interruptions as defining regions of flexibility and molecular recognition in the otherwise relatively uniform repeating collagen conformation.     

The specific recognition of a cell binding sequence derived from type I collagen by Hep3B and L929 cells

In this study, the affinity of two different cell types toward a specific cell binding sequence (Gly-Phe-Hyp-Gly-Glu-Arg or GFOGER) derived from type I collagen using peptide template (PT)-assembled collagen peptides of different triple helicity as a model for natural collagen is examined. A series of biophysical studies, including melting curve analysis and circular dichroism spectroscopy, demonstrated the presence of stable triple-helical conformation in the PT-assembled (GPO)3-GFOGER-(GPO)3, (GPO)-GFOGER-(GPO), and (Pro-Hyp-Gly)5 solution. Conversely, non-templated peptides, except (GPO)3-GFOGER-(GPO)3, showed no evidence of assembly into triple-helical structure. Biological assays, including cell adhesion, competitive inhibition, and immunofluorescence staining, revealed a correlation of triple-helical conformation with the cellular recognition of GFOGER in an integrin-specific manner. The triple helix was shown to be important, but not crucial for cell adhesion to native collagen. Hep3B and L929 cells displayed significant differences in the recognition of GFOGER, mainly because of the differences in their expression of specific integrin receptors for collagen. For example, PT-assembled (GPO)3-GFOGER-(GPO)3 was shown to perform comparably to collagen for L929, but not Hep3B, cell adhesion. The result showed that a specific cell binding motif may not fully mimic the extracellular matrix (ECM) microenvironment, suggesting the need to use a combination of two or more cell binding sequences for targeting a wide range of integrin receptors expressed by a specific cell type to better mimic the ECM.     

Folding of collagen IV

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.     

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.     

Structural Aspects of Hydroxyproline-Containing Proteins

Abstract

The occurrence of hydroxyproline (Hyp) in collagen, Clq and acetylcholinesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the β-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific β-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to Clq and acetylcholineesterase both of which share the general structural and functional properties of collagen in their “tail” regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of Clq and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Structural aspects of hydroxyproline-containing proteins

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.     

Two different heparin-binding domains in the triple-helical domain of ColQ,the collagen tail subunit of synaptic acetylcholinesterase

ColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparin-binding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina.     

Collagen model peptides: Sequence dependence of triple-helix stability

The triple helix is a specialized protein motif, found in all collagens as well as in noncollagenous proteins involved in host defense. Peptides will adopt a triple-helical conformation if the sequence contains its characteristic features of Gly as every third residue and a high content of Pro and Hyp residues. Such model peptides have proved amenable to structural studies by x-ray crystallography and NMR spectroscopy, suitable for thermodynamic and kinetic analysis, and a valuable tool in characterizing the binding activities of the collagen triple helix. A systematic approach to understanding the amino acid sequence dependence of the collagen triple helix has been initiated, based on a set of host-guest peptides of the form, (Gly-Pro-Hyp)(3)-Gly-X-Y-(Gly-Pro-Hyp)(4). Comparison of their thermal stabilities has led to a propensity scale for the X and Y positions, and the additivity of contributions of individual residues is now under investigation. The local and global stability of the collagen triple helix is normally modulated by the residues in the X and Y positions, with every third position occupied by Gly in fibril-forming collagens. However, in collagen diseases, such as osteogenesis imperfecta, a single Gly may be substituted by another residue. Host-guest studies where the Gly is replaced by various amino acids suggest that the identity of the residue in the Gly position affects the degree of destabilization and the clinical severity of the disease.