Conformational stability of type I collagen triple helix: Evidence for temporary and local relaxation of the protein conformation using a proteolytic probe

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helixto-coil transition between 25 and 42°C as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3°C lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the fulllength molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3°C lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen Nproteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35°C that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the ␣1(II) chain.The fibrillar collagens are major structural proteins that largely define the size, shape, and strength of tissues in most complex organisms (1-4). In addition, collagen fibrils are encountered in unusual biological situations such as the byssal threads whereby mussels adhere to solid surfaces (5) and in tube worms that live near deep sea hydrothermal vents (6). Collagen fibrils are formed by the spontaneous self-assembly of collagen monomers (1-4). The monomers of the major fibrillar collagens, types I, II and III, are very similar in structure in that they consist of 338 or 343 consecutive repeating tripeptide sequences of -Gly-Xxx-Yyy-tripeptide units. The -Xxx-position of the sequence is frequently proline and the -Yyy-position is frequently hydroxyproline. The tripeptide sequences with proline in the Xxx position and hydroxyproline in the Yyy position drive folding of the polypeptide chains into a unique triplehelical conformation of collagen. Other amino acids between the obligate glycine residues form hydrophobic or hydrophilic clusters on the surface of the molecules that direct the selfassembly of the proteins into stable fibrils. Because of the constraints conferred by the triple-helical conformation, the monomers of collagens are generally regarded as rigid rods. However, there are many indications that different regions of the collagen triple-helix vary in stability and undergo microunfolding in the physiological range of temperatures (7-16). Evidence for the microunfolding of the monomer included the effects of partial...

Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

Recombinant Procollagen II: Deletion of D Period Segments Identifies Sequences That Are Required for Helix Stabilization and Generates a Temperature-sensitive N-Proteinase Cleavage Site

Arnold¹,

Fertala²,

Sieroń³

et al. 1998

“…Molecular dynamics simulations indicate microunfolding of interstitial collagens at the MMP cleavage site (40 -42). Based on enzyme susceptibility, the type I collagen MMP cleavage site undergoes local reversible relaxation (43), whereas the cleavage site in type III collagen has been proposed to be more flexible than the one in type I collagen (19,44).…”

Section: Unique Features Of Interstitial Collagen Cleavage Sitesmentioning

confidence: 99%

Interstitial Collagen Catabolism

Fields

2013

167

135

Interstitial collagen mechanical and biological properties are altered by proteases that catalyze the hydrolysis of the collagen triple-helical structure. Collagenolysis is critical in development and homeostasis but also contributes to numerous pathologies. Mammalian collagenolytic enzymes include matrix metalloproteinases, cathepsin K, and neutrophil elastase, and a variety of invertebrates and pathogens possess collagenolytic enzymes. Components of the mechanism of action for the collagenolytic enzyme MMP-1 have been defined experimentally, and insights into other collagenolytic mechanisms have been provided. Ancillary biomolecules may modulate the action of collagenolytic enzymes.

“…The more labile regions may exist in a loose conformation, constantly undergoing unfolding/refolding transitions while more stable "clamp" regions prevent unfolding of the whole molecule (3, 9 -12). Such structural and dynamic heterogeneity is believed to play an important role in self-assembly (9,13) and function (3) of collagen fibers.…”

mentioning

confidence: 99%

“…For example, some flexible sites were localized by observing triple helix bending in electron microscopy (14) and/or increased susceptibility to proteolytic cleavage (9,15). Genetically generated reshuffling of different triple helix regions was shown to have a significant effect on the overall stability of the molecule (11,16).…”

mentioning

confidence: 99%

Structural Heterogeneity of Type I Collagen Triple Helix and Its Role in Osteogenesis Imperfecta

Makareeva

Mertz

Kuznetsova

et al. 2008

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (⌬T m ) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of ⌬T m with the identity of the substituting residue. Instead, we observed regular variations in ⌬T m with the substitution location in different triple helix regions. To relate the ⌬T m map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (⌬G ‡ ) from the reported peptide data. We constructed the ⌬G ‡ map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the ⌬T m and ⌬G ‡ maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the ⌬T m map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the ␣1(I) chain.The mature type I collagen molecule is a 300-nm-long triple helix formed by two ␣1(I) and one ␣2(I) chains, which is flanked by short terminal peptides. Based on the (Gly-Xaa-Yaa) 338 triplet repeat within each chain (1), the triple helix is commonly viewed as a single domain (Xaa and Yaa stand for variable residues). However, this picture may not accurately represent variations in the stability of different regions within the triple helix that fold and unfold cooperatively (2-4). The triple helix is only metastable or marginally stable at physiological temperature (3, 5-8). Its local structure appears to be highly dynamic and intimately related to local stability. The more labile regions may exist in a loose conformation, constantly undergoing unfolding/refolding transitions while more stable "clamp" regions prevent unfolding of the whole molecule (3, 9 -12). Such structural and dynamic heterogeneity is believed to play an important role in self-assembly (9, 13) and function (3) of collagen fibers.Significant progress in understanding regional variations in the triple helix stability in different collagens has been reported in recent years. For example, some flexible sites were localized by observing triple helix bending in electron microscopy (14) and/or increased susceptibility to proteolytic cleavage (9, 15). Genetically generated reshuffling of different triple helix regions was shown to have a significant effect on the overall stability of the molecule (11, 16). Relative local stability maps were proposed based on scoring different sequences (17) or on the denaturation temperature (T m ) measured for triple-helical host-guest peptides (18).The existence of looser, less stable, and tighter, more stable structural regions within the triple helix is now commonly accepted. However, knowledge of their loca...