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.     

Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.     

In vitro collagen fibril assembly in glycerol solution: evidence for a helical cooperative mechanism involving microfibrils

Glycerol inhibits the in vitro self-association of monomeric collagen into fibrils and induces the dissociation of fibrils preassembled from NaBH4-reduced collagen. These effects were investigated in an effort to understand the mechanism of fibril assembly of the protein. In PS buffer (0.03 M NaPi and 0.1 M NaCl, pH 7.0) containing 0.1-1.0 M glycerol, the self-association of type I collagen from calf skin took place only if the protein concentration was above a critical value. This critical protein concentration increased with increasing glycerol concentration. Velocity sedimentation studies showed that below the critical protein concentration and under fibril assembly conditions, the collagen was predominantly in a monomeric state. Electron microscopic examinations revealed that the collagen aggregates formed above the critical concentration consisted mostly of microfibrils of 3-5-nm diameter along with some banded fibrils were found. Collagen treated with pepsin to remove its nonhelical telopeptides also self-associated into microfibrils and fibrils in the presence of glycerol, but the reaction did not exhibit any critical concentration. These results are consistent with a mechanism of in vitro collagen fibril assembly which involves the initial formation of microfibrils through a helical cooperative mechanism. They also suggest that contacts of the nonhelical telopeptides of each collagen with its neighboring molecules provide the necessary negative free energy change for the cooperativity and that subsequent lateral association of the microfibrils leads to banded fibrils.     

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.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

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.     

NMR and CD studies of triple-helical peptides.

Triple-helix formation of the peptide (Pro-Hyp-Gly)10 was monitored by nmr and CD spectroscopy. The two-dimensional nmr spectra indicated that the Gly C alpha H and Pro C delta H proton resonances shift upfield in going from the nonhelical to helical form, while hydroxy-proline resonances are unchanged. The integrated areas of the helical and nonhelical resonances could be monitored in the one-dimensional nmr spectrum, and indicate that in the (Pro-Hyp-Gly)10 about 90% of the residues are in a defined triple-helical conformation. The introduction of a glycine to alanine substitution or the deletion of a single hydroxyproline residue in the stable triple-helical peptide (Pro-Hyp-Gly)10 still allows trimers to be formed, but the trimers show a substantial loss of triple helix and decreased thermal stability compared with (Pro-Hyp-Gly)10. Two computer models were generated for the Gly----Ala peptide, one with the Ala side chains packed inside the helix and the other with the region containing the alanines forming a beta-bend that loops out from the helix. The nmr data is more consistent with the latter model.     

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.     

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.     

Intact glycation end products containing carboxymethyl-lysine and glyoxal lysine dimer obtained from synthetic collagen model peptide

Advanced glycation end products (AGE) are accumulated in human tissues when long-lived proteins are glycated due to hyperglycemia and/or aging. In this study, we synthesized a collagen model peptide, Ac-(Pro-Hyp-Gly)(5)-Pro-Lys-Gly-(Pro-Hyp-Gly)(5)-Ala-NH(2) to investigate intact AGEs in peptides. The peptide formed a stable triple helix structure, and was subjected to glycation reactions with glucose, ribose and glyoxal. Besides carboxymethyl-lysine in the peptide, a conjugated form linked with glyoxal lysine dimer (GOLD) was detected upon treatment with glyoxal. This is the first example of intact glycation-derived dimers of peptides retaining intrinsic protein structures.     

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.     

Energetics of intrachain salt-linkage formation in collagen

The energy of formation of salt linkages between Arg or Lys with Asp or Glu in a polypeptide chain having the collagen fold have been estimated using the fully empirical energy minimization scheme AMBER. The polypeptide was considered both in an isolated and a hydrated triple helical state. The collagen fold associated with a one-bonded triple helical conformation allows intrachain salt linkages having stabilization energies of 60-100 kcal when the reacting residues are separated by no more than two intervening residues. The amino end of one side chain always approaches the carboxyl end of the other side chain, and simultaneously approaches the carbonyl oxygen of the intervening backbone residue. The salt linkage conformation and the backbone conformation of the isolated collagen fold in vacuo are maintained when the molecules are in a hydrated triple helix. These results are compatible with a fold-forming role for salt linkages, especially in proline poor regions, during collagen polypeptide synthesis, and with the persistence of intrachain salt linkages throughout molecular and fibril assembly.     

Stability and networks of hydrogen bonds of the collagen triple helical structure: influence of pH and chaotropic nature of three anions.

The thermal stability of the trimeric species formed by seven type I collagen CNBr peptides was determined at neutral and acidic pH. Melting temperature of peptide trimers and free energy change for monomer to trimer transition were used as indices of trimer stability. A greater stability at neutral pH than at acidic pH was found for all peptides analysed because in most conditions an entropic gain overwhelms an enthalpic cost. Enthalpic reasons are prevailing only in some conditions of the more acidic peptides. The overlap zone of type I collagen fibrils is more basic than the gap zone and is therefore more sensitive to variations of pH from neutral to acidic, e.g. in bone degradation when osteoclasts acidify the lacuna lying between cell and bone. Peptide trimer stability in neutral conditions is influenced also by the chaotropic nature and the concentration of three anions: chloride, sulfate and phosphate. This was more evident for sulfate at the highest concentration used (0.5 M) when a greater stability is caused by entropic reasons. The contribution of hydroxyproline to the stability of peptide trimers is greater at neutral than at acidic pH, particularly at the highest concentration of sulfate. All our data indicate that pH, chaotropic nature and concentration of three anions influence the networks of hydrogen bonds present in the collagen triple helical structure.     

Interaction of collagen molecules from the aspect of fibril formation: acid-soluble, alkali-treated, and MMP1-digested fragments of type I collagen.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37 degrees C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30 degrees C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.     

Isinglass/collagen: denaturation and functionality

Isinglass is widely used commercially to clarify alcoholic beverages by aggregation of the yeast and other insoluble particles. It is derived from swim bladders of tropical fish by solubilisation in organic acids and consists predominantly of the protein collagen. The low content of intermolecular cross-links allows ready dissolution of swim bladder compared to bovine hide which is cross-linked by a high proportion of stable bonds and requires enzymic digestion to solubilise. Isinglass is no longer effective as a clarifying agent if thermally denatured hence the collagenous triple helical structure must be maintained. Thermal denaturation of isinglass occurs at 29 degrees C, compared to 40-41 degrees C for mammalian collagens, primarily due to the lower hydroxyproline content. The hydroxyproline is essential for the formation of H-bonded water-bridges through the hydroxyl group and the peptide chain thereby stabilising the triple helix. Based on the lower enthalpy determined by differential scanning calorimetry we have calculated that the thermally labile domain of the isinglass molecule was 41 residues compared to 66 for mammalian collagen. The fining efficiency was unaffected by pH, chelating agents, detergents and removal of surface proteins from yeast cells. Studies on the mechanism of action of isinglass have shown that higher molecular weight aggregates that increase the length of the collagen molecules (trimers, tetramers, etc.) increase efficiency and that their surface charge are important in the clarification process. By chemical modification, we have shown that blocking positively charged groups had no effect on the fining process, whilst negative charges are clearly essential and that increasing the negative charge by succinylation increases its efficacy. Solutions of bovine hide collagen were shown to be equally effective in refining beers and standard yeast preparations. The higher thermal denaturation temperature, ready availability and reproducibility of bovine collagen preparations gives it considerable advantages over isinglass.     

Glycosylation/Hydroxylation-induced stabilization of the collagen triple helix. 4-trans-hydroxyproline in the Xaa position can stabilize the triple helix

We have shown recently that glycosylation of threonine in the peptide Ac-(Gly-Pro-Thr)(10)-NH(2) with beta-d-galactose induces the formation of a collagen triple helix, whereas the nonglycosylated peptide does not. In this report, we present evidence that a collagen triple helix can also be formed in the Ac-(Gly-Pro-Thr)(10)-NH(2) peptide, if the proline (Pro) in the Xaa position is replaced with 4-trans-hydroxyproline (Hyp). Furthermore, replacement of Pro with Hyp in the sequence Ac-(Gly-Pro-Thr(beta-d-Gal))(10)-NH(2) increases the T(m) of the triple helix by 15.7 degrees C. It is generally believed that Hyp in the Xaa position destabilizes the triple helix because (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) form stable triple helices but the peptide (Hyp-Pro-Gly)(10) does not. Our data suggest that the destabilizing effect of Hyp relative to Pro in the Xaa position is only true in the case of (Hyp-Pro-Gly)(10). Increasing concentrations of galactose in the solvent stabilize the triple helix of Ac-(Gly-Hyp-Thr)(10)-NH(2) but to a much lesser extent than that achieved by covalently linked galactose. The data explain some of the forces governing the stability of the annelid/vestimentiferan cuticle collagens.     

Collagens

The collagens represent a family of trimeric extracellular matrix molecules used by cells for structural integrity and other functions. The three α chains that form the triple helical part of the molecule are composed of repeating peptide triplets of glycine-X-Y. X and Y can be any amino acid but are often proline and hydroxyproline, respectively. Flanking the triple helical regions (i.e., Col domains) are non-glycine-X-Y regions, termed non-collagenous domains. These frequently contain recognizable peptide modules found in other matrix molecules. Proper tissue function depends on correctly assembled molecular aggregates being incorporated into the matrix. This review highlights some of the structural characteristics of collagen types I-XXVIII.     

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.     

Asymmetry in the triple helix of collagen-like heterotrimers confirms that external bonds stabilize collagen structure

Heating and subsequent cooling mixtures of (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) peptides leads to formation of model heterotrimeric collagen helices that can be isolated by HPLC. These heterotrimeric collagen peptide helices are shown to be fundamentally unstable as denaturing then renaturing experiments result in heterotrimeric/homotrimeric mixtures.As the proportion of hydroxyproline-containing chains in the trimers increases, differential scanning calorimetry shows that the helix melting temperatures and denaturation enthalpies increasing non-linearly. Three types of Rich-Crick hydrogen bonds observed by NMR allow modelling of heterotrimeric structures based on published homotrimeric X-ray data. This revealed a small axial movement of (Pro-Hyp-Gly)(10) chains towards the C-terminal of the helix, demonstrating heterotrimeric asymmetry.