The Self-assembly of a Mini-fibril with Axial Periodicity from a Designed Collagen-mimetic Triple Helix

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.     

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.     

The crucial role of trimerization domains in collagen folding

Collagens contain large numbers of Gly-Xaa-Yaa peptide repeats that form the characteristic triple helix, where the individual chains fold into a polyproline II helix and three of these helices form a right-handed triple helix. For the proper folding of the triple helix collagens contain trimerization domains. These domains ensure a single starting point for triple helix formation and are also responsible for the chain selection in heterotrimeric collagens. Trimerization domains are non-collagenous domains of very different structures. The size of trimerization domains varies from 35 residues in type IX collagen to around 250 residues for the fibrillar collagens. These domains are not only crucial for biological functions, but they are also attractive tools for generating recombinant collagen fragments of interest as well as for general use in protein engineering and biomaterial design. Here we review the current knowledge of the structure and function of these trimerization domains.     

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.     

2005 Emil Thomas Kaiser Award

Collagen is the most abundant protein in animals. The conformational stability of the collagen triple helix is enhanced by the hydroxyl group of its prevalent (2S,4R)-4-hydroxyproline residues. For 25 years, the prevailing paradigm had been that this enhanced stability is due to hydrogen bonds mediated by bridging water molecules. We tested this hypothesis with synthetic collagen triple helices containing 4-fluoroproline residues. The results have unveiled a wealth of stereoelectronic effects that contribute markedly to the stability of collagen, as well as other proteins. This new understanding is leading to synthetic collagens for a variety of applications in biotechnology and biomedicine.     

Amino acid sequence of the triple-helical domain of human collagen type VI

The complete amino acid sequence of the triple-helical domain of human collagen VI was deduced from sequences of appropriate cDNA clones and confirmed to about 50% by Edman degradation of tryptic peptides. This domain consists of three different peptide segments containing some 335-336 amino acid residues originating from central portions of the alpha 1 (VI), alpha 2(VI), and alpha 3(VI) chains, respectively. Sequence identity in the X/Y positions of the Gly-X-Y repeats is rather low (10-15%) between the chains. Peculiar features of these sequences include 3 cysteine residues about 50 (alpha 3(VI)) and 89 (alpha 1(VI), alpha 2(VI)) residues away from the N-terminus and several Gly-X-Y interruptions clustered in the C-terminal two-thirds of the triple helix. These structures are presumably required for cross-linking collagen VI oligomers and for super-coiling of triple helices in the dimers. Other features include 11 Arg-Gly-Asp sequences, some of which are likely to be used as cell-binding sites, and four Asn-X-Thr sequences, allowing N-linked glycosylation along the triple helix. Junctional areas close to the helix contain short, cysteine-rich segments which may seal the triple-helical domain through disulfide bond formation, endowing it with high stability. These features, together with a low sequence homology to fiber-forming and basement-membrane collagens, document the unique character of collagen VI, whose triple helix is specifically adjusted for forming microfibrils in tissues.     

Differentiation of secreted and membrane-type matrix metalloproteinase activities based on substitutions and interruptions of triple-helical sequences

The turnover of the collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology and has been ascribed to a small number of proteases. Several members of the matrix metalloproteinase (MMPs) family possess collagenolytic activity, and the mechanisms by which these enzymes process triple helices are beginning to be unraveled. The present study has utilized two triple-helical sequences to compare the cleavage-site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly approximately Met-Arg-Gly), while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn approximately Phe-Arg-Gly). Both sequences were selectively cleaved by MMP-13 while in linear form, but neither proved to be selective within a triple helix. This suggests that the conformational presentation of substrate sequences to a MMP active site is critical for enzyme specificity, in that activities differ when sequences are presented from an unwound triple helix versus an independent single strand. Differences in specificity between secreted and membrane-type (MT) MMPs were also observed for both sequences, where MMP-2 and MT-MMPs showed an ability to hydrolyze a triple helix at an additional site (Gly-Gln bond). Interruption of the triple helix had different effects on secreted MMPs and MT-MMPs, because MT-MMPs could not hydrolyze the Asn-Phe bond but instead cleaved the triple helix closer to the C terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P1 subsite to be able to efficiently process a triple-helical molecule. Analysis of individual kinetic parameters and activation energies indicated different substrate preferences within secreted MMPs, because MMP-13 preferred the interrupted sequence, while MMP-8 showed little discrimination between non-interrupted and interrupted triple helices. On the basis of the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such differences may be significant for understanding MMP mechanisms of action and aid in the development of selective MMP inhibitors.     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.     

Computation of 3D structure and distribution of helical parameters for D-period segments of human fibrillar collagen III

The structures of D-period segments of collagen (234 amino residues or ～1/4 of whole length) are established by methods of molecular mechanics and geometry analysis. Each D-period segment proves to have a unique spatial structure. The distributions of local helical parameters along the molecule are calculated. It is found that a second hydrogen bond is formed in every case when the second residue in the tripeptide G-X-Y is an amino acid. With such a combined H-bond network, all the peptide CO groups of glycines and of the third residues in tripeptides have quasi-equivalent positions on the surface of the collagen molecule. The local deformations of the polyproline II helix in the triple complex give rise to the observed modulation of structure at the macromolecular level, which may be important for the mutual orientation of collagen molecules during fibrillogenesis.     

Chirality‐specific lift forces of helix under shear flows: Helix perpendicular to shear plane

Chiral objects in shear flow experience a chirality‐specific lift force. Shear flows past helices in a low Reynolds number regime were studied using slender‐body theory. The chirality‐specific lift forces in the vorticity direction experienced by helices are dominated by a set of helix geometry parameters: helix radius, pitch length, number of turns, and helix phase angle. Its analytical formula is given. The chirality‐specific forces are the physical reasons for the chiral separation of helices in shear flow. Our results are well supported by the latest experimental observations.     

4-chloroprolines: synthesis, conformational analysis, and effect on the collagen triple helix

Collagen is an abundant, triple-helical protein comprising three strands of the repeating sequence: Xaa-Yaa-Gly. (2S)-Proline and (2S,4R)-4-hydroxyproline (Hyp) are common in the primary structure of collagen. Here, we use nonnatural proline derivatives to reveal determinants of collagen stability. Specifically, we report high-yielding syntheses of (2S,4S)-4-chloroproline (clp) and (2S,4R)-4-chloroproline (Clp). We find that the molecular structure of Ac-Clp-OMe in the solid state is virtually identical to that of Ac-Hyp-OMe. In contrast, the conformational properties of Ac-clp-OMe are similar to those of Ac-Pro-OMe. Ac-Clp-OMe has a stronger preference for a trans amide bond than does Ac-Pro-OMe, whereas Ac-clp-OMe has a weaker preference. (Pro-Clp-Gly)(10) forms triple helices that are significantly more stable than those of (Pro-Pro-Gly)(10). Triple helices of (clp-Pro-Gly)(10) have stability similar to those of (Pro-Pro-Gly)(10). Unlike (Pro-Clp-Gly)(10) and (clp-Pro-Gly)(10), (clp-Clp-Gly)(10) does not form a stable triple helix, presumably due to a deleterious steric interaction between proximal chlorines on different strands. These data, which are consistent with previous work on 4-fluoroprolines and 4-methylprolines, support the importance of stereoelectronic and steric effects in the stability of the collagen triple helix and provide another means to modulate that stability. (     

Structural hierarchy controls deformation behavior of collagen

The structure of collagen, the most abundant protein in mammals, consists of a triple helix composed of three helical polypeptide chains. The deformation behavior of collagen is governed by molecular mechanisms that involve the interaction between different helical hierarchies found in collagen. Here, we report results of Steered Molecular Dynamics study of the full-length collagen molecule (～290 nm). The collagen molecule is extended at various pulling rates ranging from 0.00003/ps to 0.012/ps. These simulations reveal a new level of hierarchy exhibited by collagen: helicity of the triple chain. This level of hierarchy is apparent at the 290 nm length and cannot be observed in the 7-9 nm models often described to evaluate collagen mechanics. The deformation mechanisms in collagen are governed by all three levels of hierarchy, helicity of single chain (level-1), helical triple helix (level-2), and hereby described helicity of the triple chain (level-3). The mechanics resulting from the three levels is described by an interlocking gear analogy. In addition, remarkably, the full-length collagen does not show much unwinding of triple helix unlike that exhibited by short collagen models. Further, the full-length collagen does not show significant unwinding of the triple helix, unlike that exhibited by short collagen. Also reported is that the interchain hydrogen bond energy in the full-length collagen is significantly smaller than the overall interchain nonbonded interaction energies, suggesting that the nonbonded interactions have far more important role than hydrogen bonds in the mechanics of collagen. However, hydrogen bonding is essential for the triple helical conformation of the collagen. Hence, although mechanics of collagen is controlled by nonbonded interchain interaction energies, the confirmation of collagen is attributed to the interchain hydrogen bonding.     

Contributions of cation-π interactions to the collagen triple helix stability

Cation–π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X–Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation–π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host–guest peptide system: (Pro-Hyp-Gly)₃-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)₃, where X is an aromatic residue and Y is a cationic residue, to study the cation–π interaction in the collagen triple helix. Circular dichroism (CD) measurements and T_m data analysis show that the cation–π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation–π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation–π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π–electron density and the cation–π interaction in collagen. Our data demonstrate that the cation–π interaction could play an important role in stabilizing the collagen triple helix.     

Crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly at 1.4 A resolution: implications for collagen hydration

The use of polypeptide models has proved to be a valuable tool to obtain accurate information on the collagen triple helix. Here we report the high resolution crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly. The structure has been refined to an R(factor) of 0.137 and an R(free) of 0.163 using synchrotron diffraction data extending up to 1.4 A resolution. The polypeptide triple-helical structure binds a large number of water molecules, in contrast with a previous structure determination at lower resolution. The highly hydrated nature of this polypeptide confirms a number of previous studies conducted both in solution and in the crystal state. In addition, neighboring polypeptide triple helices are directly bound in the crystal through Hyp-Hyp hydrogen-bonding interactions. This finding supports the idea that Hyp residues may be important for the assembly of the triple helices in the collagen fibrils and may stabilize the fibrils by mediating direct contacts between neighboring molecules.     

Helix-specific interactions induce condensation of guanosine four-stranded helices in concentrated salt solutions.

Deoxyguanosine-5'-monophosphate in water self-associates into stable structures, which include liquid-crystalline hexagonal and cholesteric phases. The structural unit is a four-stranded helix, composed of stacked Hoogsteen-bonded guanosine quartets. By using the osmotic stress method, we recently measured the force between helices in KCl solutions up to 2 M. In addition to the long-range electrostatic force, a short-range hydration repulsive contribution was recognized. The hydration repulsion is exponential, and shows a decay length independent from the ionic strength of the solution. Here, we report that more concentrated KCl solutions cause condensation of the guanosine helix in a hexagonal phase with constant equilibrium separation of approximately 7 A between helix surfaces. Long-range attraction, which induces the self-assembly, and short-range repulsion, which prevents the contact between the helices, are implied. By using osmotic stress, the force needed to push helices closer from the spontaneously assumed position has been measured. The attractive force was then estimated as a difference between the net force and the repulsive contribution, revealing an exponential decay length about two times larger than that of the short-range repulsion. The agreement with the helix interaction theory introduced recently by Kornyshev and Leikin (Kornyshev, A. A., and S. Leikin, 1997. Theory of interaction between helical molecules. J. Phys. Chem. 107:3656-3674) suggests that the repulsive and attractive forces originate from helix-specific interactions.     

Crosslinking between alpha and beta subunits defines the orientation and spatial relationship of some of the transmembrane helices of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli

The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and either eight or nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1, 10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the beta subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydstr?m, J. Biol. Chem. 274, 19072-19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the alpha subunit are close to helix 7. In the alpha(2)beta(2) tetramer, helices 2 and 4 of one alpha subunit are close to the same pair of transmembrane helices of the other alpha subunit, and helix 6 of one beta subunit is close to helix 6 of the other beta subunit.     

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by B?chinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.     

Mechanical properties of physiological and pathological models of collagen peptides investigated via steered molecular dynamics simulations

In this work we used molecular simulations to investigate the elastic properties of collagen single chain and triple helix with the aim of understanding its features starting from first principles. We analysed ideal collagen peptides, homotrimeric and heterotrimeric collagen type I and pathological models of collagen. Triple helices were found much more rigid than single chains, thus enlightening the important role of interchain stabilizing forces, like hydrophobic interaction and hydrogen bonds. We obtained Young's moduli close to 4.5GPa for the ideal model of collagen and for the physiological heterotrimer, while the physiological homotrimer presented a Young's modulus of 2.51GPa, that can be related to a mild form of Osteogenesis Imperfecta in which only the homotrimeric form of collagen type I is produced. Otherwise, the pathological model (presenting a glycine to alanine substitution) showed an elastic modulus of 4.32GPa, thus only slightly lower than the ideal model. This suggests that this mutation only slightly affects the mechanical properties of the collagen molecule, but possibly acts on an higher scale, such as the packing of collagen fibrils.     

Hydroxylation-induced stabilization of the collagen triple helix. Further characterization of peptides with 4(R)-hydroxyproline in the Xaa position

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 degrees 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.     

Crystal structure of human type III collagen Gly991-Gly1032 cystine knot-containing peptide shows both 7/2 and 10/3 triple helical symmetries

Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)(n) or (Gly-Pro-Hyp)(n). To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly(991)-Gly(1032) structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.