Stereochemistry of collagen

This review article, based on a lecture delivered in Madras in 1985, is an account of the author's experience in the working out of the molecular structure and conformation of the collagen triple-helix over the years 1952-78. It starts with the first proposal of the correct triple-helix in 1954, but with three residues per turn, which was later refined in 1955 into a coiled-coil structure with approximately 3.3 residues per turn. The structure readily fitted proline and hydroxyproline residues and required glycine as every third residue in each of the three chains. The controversy regarding the number of hydrogen bonds per tripeptide could not be resolved by X-ray diffraction or energy minimization, but physicochemical data, obtained in other laboratories during 1961-65, strongly pointed to two hydrogen bonds, as suggested by the author. However, it was felt that the structure with one straight NH...O bond was better. A reconciliation of the two was obtained in Chicago in 1968, by showing that the second hydrogen bond is via a water molecule, which makes it weaker, as found in the physicochemical studies mentioned above. This water molecule was also shown, in 1973, to take part in further cross-linking hydrogen bonds with the OH group of hydroxyproline, which occurred always in the location previous to glycine, and is at the right distance from the water. Thus, almost all features of the primary structure, X-ray pattern, optical and hydrodynamic data, and the role of hydroxyproline in stabilising the triple helical structure, have been satisfactorily accounted for. These also lead to a confirmation of Pauling's theory that vitamin C improves immunity to diseases, as explained in the last section.     

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.     

Structure of collagen with a new net of hydrogen bonds]

A conformational variability of the collagen triple helix was studied with the methods of molecular mechanics. The Rich-Crick model with one hydrogen bond per tripeptide fragment or the model with two hydrogen bonds per tripeptide fragment were used for tripeptides forming the primary structure of the protein. Imino acid and amino acid residues were located in the second position of the tripeptide fragments in the first and second cases, respectively. Conformations on domain boundaries, which had alternating structures with one and two hydrogen bonds per tripeptide, were particularly studied. Essentially all types of collagen backbone composed of amino acid residues most frequently occurring in this protein were considered. A new model was suggested that combined elements of the Rich-Crick model and our new approach. This was shown to be stereochemically valid, energetically advantageous, and consistent with the experimental data. It was conclusively demonstrated that the primary structure of collagen determines its tertiary structure.     

A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

A triple-helical model for (Gly-Pro-Hyp)n with cis peptide units

Synthetic regular polytripeptides of the type (Gly-R₂-R₃) where R₂, R₃, or both, are imino acids have been widely studied as model compounds for collagen. One such polytripeptide is poly(Gly-Pro-Hyp), since triplets with this sequence constitute about 10% of collagen. Recently, a new model has been proposed for this polytripeptide in which one of the three peptide bonds in the tripeptide unit is in the cis conformation, and the γ-hydroxyl group of hydroxyproline forms a direct interchain hydrogen bond within the triple helix. We have confirmed this structure by model building using computer techniques, and the helical parameters obtained by us are close to the experimentally observed values. The model is also found to be comparable in stability with other models from energy considerations.     

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.     

Stabilization of collagen fibrils by hydroxyproline

The substitution of hydroxyproline for proline in position Y of the repeating Gly-X-Y tripeptide sequence of collagen-like poly(tripeptide)s (i.e., in the position in which Hyp occurs naturally) is predicted to enhance the stability of aggregates of triple helices, while the substitution of Hyp in position X (where no Hyp occurs naturally) is predicted to decrease the stability of aggregates. Earlier conformational energy computations have indicated that two triple helices composed of poly(Gly-Pro-Pro) polypeptide chains pack preferentially with a nearly parallel orientation of the helix axes [Nemethy, G., & Scheraga, H.A. (1984) Biopolymers 23, 2781-2799]. Conformational energy computations reported here indicate that the same packing arrangement is preferred for the packing of two poly(Gly-Pro-Hyp) triple helices. The OH groups of the Hyp residues can be accommodated in the space between the two packed triple helices without any steric hindrance. They actually contribute about 1.9 kcal/mol per Gly-Pro-Hyp tripeptide to the packing energy, as a result of the formation of weak hydrogen bonds and other favorable noncovalent interatomic interactions. On the other hand, the substitution of Hyp in position X weakens the packing by about 1.7 kcal/mol per Gly-Hyp-Pro tripeptide. Numerous published experimental studies have established that Hyp in position Y stabilizes an isolated triple helix relative to dissociated random coils, while Hyp in position X has the opposite effect. We propose that Hyp in position Y also enhances the stability of the assembly of collagen into microfibrils while, in position X, it decreases this stability.     

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.     

Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers.

Collagen molecules in solution unfold close to the maximum body temperature of the species of animal from which the molecules are extracted. It is therefore vital that collagen is stabilized during fiber formation. In this paper, our concept that the collagen molecule is thermally stabilized by loss of configurational entropy of the molecule in the fiber lattice, is refined by examining the process theoretically. Combining an equation for the entropy of a polymer-in-a-box with our previously published rate theory analysis of collagen denaturation, we have derived a hyperbolic relationship between the denaturation temperature, Tm, and the volume fraction, epsilon, of water in the fiber. DSC data were consistent with the model for water volume fractions greater than 0.2. At a water volume fraction of about 0.2, there was an abrupt change in the slope of the linear relationship between 1/Tm and epsilon. This may have been caused by a collapse of the gap-overlap fiber structure at low hydrations. At more than 6 moles water per tripeptide, the enthalpy of denaturation on a dry tendon basis was independent of hydration at 58.55 +/- 0.59 J g-1. Between about 6 and 1 moles water per tripeptide, dehydration caused a substantial loss of enthalpy of denaturation, caused by a loss of water bridges from the hydration network surrounding the triple helix. At very low hydrations (less than 1 mole of water per tripeptide), where there was not enough water to form bridges and only sufficient to hydrogen bond to primary binding sites on the peptide chains, the enthalpy was approximately constant at 11.6 +/- 0.69 J g-1. This was assigned mainly to the breaking of the direct hydrogen bonds between the alpha chains.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Triple helical structure and stabilization of collagen-like molecules with 4(R)-hydroxyproline in the Xaa position

In this study, we examine the relationships between the structure and stability of five related collagen-like molecules that have hydroxyproline residues occupying positions not observed in vertebrate collagen. Two of the molecules contain valine or threonine and form stable triple helices in water. Three of the molecules contain allo-threonine (an enantiomer of threonine), serine, or alanine, and are not stable. Using molecular dynamics simulation methods, we examine possible explanations for the stability difference, including considering the possibility that differences in solvent shielding of the essential interchain hydrogen bonds may result in differences in stability. By comparing the structures of threonine- and allo-threonine-containing molecules in six polar and nonpolar solvation conditions, we find that solvent shielding is not an adequate explanation for the stability difference. A closer examination of the peptides shows that the structures of the unstable molecules are looser, having weaker intermolecular hydrogen bonds. The weakened hydrogen bonds result from extended Yaa residue Psi-angles that prevent optimal geometry. The Phi-Psi-maps of the relevant residues suggest that each residue's most favorable Psi-angle determines the corresponding collagen-like molecule's stability. Additionally, we propose that these molecules illustrate a more general feature of triple-helical structures: interchain hydrogen bonds are always longer and weaker than ideal, so they are sensitive to relatively small changes in molecular structure. This sensitivity to small changes may explain why large stability differences often result from seemingly small changes in residue sequence.     

Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogenous peptides resembling collagen.

To explore further the recent demonstration that hydroxyproline stabilizes the triple-helical structure of collagen, two peptides containing allohydroxyproline, (aHyp-Pro-Gly)10 and (Pro-aHyp-Gly)10, were synthesized by a modified Merrifield technique which yields products of defined molecular weight. Examination of the peptides by optical rotation and circular dichroism showed that neither of them formed triple-helical structures in aqueous solution. Since the peptides had less tendency than (Pro-Hyp-Gly)10 to become helical, the results demonstrated that the trans-4-hydroxyl group of hydroxyproline makes a specific contribution to stability of the triple helix formed by (Pro-Hyp-Gly)10. Since the peptides also had less tendency than (Pro-Pro-Gly10 to become helical, the results further demonstrated that the cis-4-hydroxyl group on allohydroxyproline decreases the stability of the triple helix. The observations provided direct support for previous data indicating that incorporation of proline analogues such as allohydroxyproline into pro-alpha chains during procollagen biosynthesis prevents the polypeptides from becoming triple helical.     

Helical conformations of three crystalline pentapeptide fragments of suzukacillin,a membrane channel forming polypeptide

The crystal structures of three pentapeptide fragments of suzukacillin-A have been determined. Boc-Aib-Pro-Val-Aib-Val-OMe (peptide 1–5) adopts a distorted helical conformation, stabilized by three intramolecular hydrogen bonds (two 5→1, one 4→1). Boc-Ala-Aib-Ala-Aib-Aib-OMe (peptide 6–10) and Boc-Leu-Aib-Pro-Val-Aib-OMe (peptide 16–20) adopt 3₁₀ helical structures stabilized by three and two 4→1 intramolecular hydrogen bonds, respectively. These structures provide substantial support for a largely helical conformation for the suzukacillin membrane channel.     

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.     

Self‐association of streptococcus pyogenes collagen‐like constructs into higher order structures

A number of bacterial collagen‐like proteins with Gly as every third residue and a high Pro content have been observed to form stable triple‐helical structures despite the absence of hydroxyproline (Hyp). Here, the high yield cold‐shock expression system is used to obtain purified recombinant collagen‐like protein (V‐CL) from Streptococcus pyogenes containing an N‐terminal globular domain V followed by the collagen triple‐helix domain CL and the modified construct with two tandem collagen domains V‐CL‐CL. Both constructs and their isolated collagenous domains form stable triple‐helices characterized by very sharp thermal transitions at 35–37°C and by high values of calorimetric enthalpy. Procedures for the formation of collagen SLS crystallites lead to parallel arrays of in register V‐CL‐CL molecules, as well as centrosymmetric arrays of dimers joined at their globular domains. At neutral pH and high concentrations, the bacterial constructs all show a tendency towards aggregation. The isolated collagen domains, CL and CL‐CL, form units of diameter 4–5 nm which bundle together and twist to make larger fibrillar structures. Thus, although this S. pyogenes collagen‐like protein is a cell surface protein with no indication of participation in higher order structure, the triple‐helix domain has the potential of forming fibrillar structures even in the absence of hydroxyproline. The formation of fibrils suggests bacterial collagen proteins may be useful for biomaterials and tissue engineering applications.     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

Roles of collagen fibers and its specific molecular chaperone: analysis using HSP47-knockout mice.

Collagen is the most abundant protein of mammals and produces highly organized ultrastructures in the extracellular matrix. There are at least 27 types of collagen in mammalian tissues. While fibrillar collagen (eg. types I, II, III, V and XI) assembles into large fibril structures in the extracellular matrix, type IV collagen produces meshwork-like structures in the basement membranes. As collagen has a distinct triple helix structure composed of Gly-X-Y repeats whose Y position is often hydroxyproline, its folding and maturation process differs considerably from globular proteins. Type I collagen is an assembly of two alpha-1 chains and one alpha-2 chain, and each of the alpha chains contain the N-terminal propeptide, C-terminal propeptide and central triple helical region. The 47-kDa heat shock protein (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes the triple helical region of collagen and is required for productive folding and maturation of collagen molecules. Only in the presence of HSP47, collagen type I molecules can be assembled into the correctly folded triple helices in the ER of mouse embryos without producing misfolded or non-functionally aggregated molecules. HSP47-knockout embryos die just after 10.5 day due to the absence of functional collagen. Recent our data demonstrated that the non-fibrillar network-forming collagen type IV also requires HSP47 for productive folding and maturation. Here, we discuss the role of HSP47 in the folding and maturation of collagen type IV as well as type I.     

Structural analysis of peptide helices containing centrally positioned lactic acid residues

The effect of insertion of lactic acid (Lac) residues into peptide helices has been probed using specifically designed sequences. The crystal structures of 11-residue and 14-residue depsipeptides Boc-Val-Val-Ala-Leu-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (1) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (3), containing centrally positioned Lac residues, have been determined. The structure of an 11-residue peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (2), analog of a which is an amide previously determined Lac-containing depsipeptide, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe I. L. Karle, C. Das, and P. Balaram, Biopolymers, Vol. 59, (2001) pp. 276-289], is also reported. Peptide 1 adopts a helical fold, which is stabilized by mixture of 4-->1 and 5-->1 hydrogen bonds. Peptide 2 adopts a completely alpha-helical conformation stabilized by eight successive 5-->1 hydrogen bonds. Peptide 3 appears to be predominately alpha-helical, with seven 5-->1 hydrogen bonds and three 4-->1 interaction interspersed in the sequence. In the structure of peptide 3 in addition to water molecules in the head-to-tail region, hydration at an internal segment of the helix is also observed. A comparison of five related peptide helices, containing a single Lac residue, reveals that the hydroxy acid can be comfortably accommodated at interior positions in the helix, with the closest C=O...O distances lying between 2.8 and 3.3 A.     

Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.     

Self-association of collagen triple helic peptides into higher order structures

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.