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Threonine in Collagen Triple-helical Structure



Crystallization of theronine (Thr) peptide in collagen triple-helical structure was investigated. The crystallization process was performed by hanging drop-diffusion method at 4°C and sample solution was prepared at concentrated 10 mg mL-1. Rod-like crystals appeared in three weeks and a single crystal was measured at 100 K on the BBL6A beamline at the Photon factory in Tsukuba. The results indicated that the hydroxide group (OH) of theronine in the Yaa position participate in water-mediated inter and intra-chain hydrogen bonds in the similar way to the OH group of 4(R)Hyp. Only T3-78515 has reported a structure of Thr in a triple-helical structure.
Threonine in Collagen Triple-helical Structure
Nattha JIRAVANICHANUN,1;2Kazunori MIZUNO,3Hans Peter BA
1Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan
2Department of Biotechnology and Life Science, Graduate School of Engineering,
Tokyo University of Agriculture and Technology, Koganei 184-8588, Japan
3Department of Biochemistry and Molecular Biology, Oregon Health & Science University,
and Shriners Hospital for Children, Research Department, Portland, Oregon 97239, USA
(Received October 4, 2005; Accepted December 5, 2005; Published April 15, 2006)
KEY WORDS Threonine / Collagen / Triple-helical Structure / Host-guest Peptide / Side-chain
Conformation / Hydration Pattern /
[DOI 10.1295/polymj.38.400]
Collagen is the most abundant proteins found in the
extracellular matrix of multicellular animals, and has
a unique triple-helical structure, which is composed
of the three polypeptides. The three chains form a
right-handed supercoiled triple-helix. Each polypep-
tide chain requires Gly at every third residue, which
generates -Xaa-Yaa-Gly- repeating sequence. The
glycine residues in every third position are packed
in the center of the triple-helix. The residues in the
Xaa and the Yaa positions are exposed to the molecu-
lar surface. High contents of imino acids in the Xaa
and the Yaa positions are required to the stability of
the structure. Collagen family includes more than
thirty proteins in vertebrates.
Similar collagens and
much more diverse collagen proteins are also present-
ed throughout invertebrates including a few giant
molecules found in the cuticles of several worm spe-
For example, the Riftia pachyptila cuticle col-
lagen has a low Hyp content but the Thr content is
much higher than those found in other collagens.
The mechanism of the stability of the collagen helix
is still unknown. The Thr of the collagen is highly gly-
Several model peptides were synthesized
to analyze its thermal stability and property.
O-galactosylation of Thr increases the thermal stability
(the helix-coil transition temperature) of Ac-(Gly-Pro/
The CD experiments
of Ac-(Gly-4(R)Hyp-Yaa)10-NH2peptides with vari-
ous amino acids in the Yaa position (Thr, Ser, Val,
Ala, and alloThr) suggested that the methyl group,
hydroxyl group and stereo configuration of Thr are im-
portant for the stability.
The methyl group of Thr was
hypothesized to shield the inter-chain hydrogen bond
between the amide of Gly and carbonyl of Xaa resi-
dues from water molecules by energy-minimization
Although several studies have challenged
to rationalize the experimental data, the mechanism
of the stability in cuticle collagen is still ambiguous.
In order to understand the stabilization mechanism
of Thr in the Yaa position, we attempted to crystallize
the peptides Ac-(Gly-4(R)Hyp-Thr)10-NH2and H-
(Gly-4(R)Hyp-Thr)10-OH. Despite their ability to
form a triple-helical structure,
we could not succeed
in the formation of the single crystals of these peptides
yet. The host-guest peptide system is an alternative
way to get single crystals of the peptide with interest-
ing sequence. Therefore, 4(R)Hyp-Thr-Gly tripeptide
unit was inserted into the stable host peptide (Pro-Pro-
The host-guest peptide H-(Pro-Pro-Gly)4-
(4(R)Hyp-Thr-Gly)-(Pro-Pro-Gly)4-OH (OTG) con-
tains 4(R)Hyp-Thr-Gly tripeptide unit that is abundant
in the Riftia pachyptila cuticle collagen. The single
crystal analysis of the OTG peptide provided the first
insight into the unique 4(R)Hyp-Thr-Gly tripeptide
unit conformation. Here, the Thr conformation and
the observed hydration patterns around Thr residue
in triple-helical structure were revealed.
Crystallization was performed by hanging-drop dif-
fusion method at 4 C. Sample solution was prepared
at concentrated 10 mg mL1. Reservoir solution con-
tained 0.1 M Hepes buffer pH 7.5 and 23% (w/v)
PEG1000. Drop mixture made up of sample solution
2ml and reservoir solution 2 ml. Rod-like crystals ap-
peared in about 3 weeks. A single crystal was meas-
ured at 100 K on the beamline BL6A at the Photon
Factory in Tsukuba. Intensity data was processed by
Crystal belongs to monoclinic space
To whom correspondence should be addressed (Tel: +81-66-850-5455, Fax: +81-66-850-5455, E-mail:
Polymer Journal, Vol. 38, No. 4, pp. 400–403 (2006)
group P21with unit cell parameters a¼26:0,b¼
˚,¼90:4. The structure of OTG
was determined by molecular replacement method
using (Pro-Pro-Gly)9peptide (PDB code 1ITT)
a search model. Positional refinement was performed
and structure refinement was carried
out by SHELX-L.
Thr residues are at the central tripeptide unit of the
molecule. To describe side-chain conformation of
Thr, 1dihedral angle is defined by N-C-C-O1or
N-C-C-C2. The different conformations of the
side-chain as a function of 1values of 60, 180,
and 60are referred to gaucheþ,trans, and gauche,
respectively. Thus, Thr side-chain in OTG structure,
the O1takes gaucheþconformation, whereas the
C2takes trans conformation to amide group (Figure
1). Both the O1and the C2are directed toward adja-
cent chains. This kind of Thr side-chain conformation
is the same as two out of three Thr in T3-785 pep-
The average main-chain dihedral angles (= )
of Thr in this study are 61and 145, which are con-
sistent with those values in the Yaa position of colla-
gen-like peptides.
In T3-785 structure,
mediated hydrogen bond was reported between the
Thr OH group and the Gly carbonyl in the same chain
via one water molecule. For Thr, not only the above
water-mediated pattern, but also diverse water-medi-
ated patterns are observed in the OTG structure in
Figure 2a. In the first case, two water molecules make
hydrogen bonds with the OH group of Thr114 and the
Figure 1. gaucheþ/trans conformation of Thr in the OTG
Gly1A O
Gly2B O
Gly112 O
Thr314 O 1γ
Hyp3A O
Thr114 N Hyp4B Oδ
Thr114 O 1
Hyp5C O
Hyp4B O
Thr114 O Thr214 O 1
Hyp5C O
Pro317 O
Hyp6A O
(a) (b)
Figure 2. (a) Central region of the OTG molecule shows three cases of water-mediated hydrogen bonds at OH group of Thr. Case 1:
Thr114 O1connects through two water molecules to Gly112 O in the same chain. Case 2: Thr114 O1connects through two water mole-
cules to Thr114 N within the same residue. Case 3: Thr314 O1connects through two water molecules to Thr114 O of adjacent chain and
Thr214 O1connects through two water molecules to Pro317 O of adjacent chain. In the residue name, the first digit corresponds to a chain
number and the next two digits correspond to a residue number. (b) Hydration patterns involving OH group of 4(R)Hyp and carbonyl
groups in (Pro-4(R)Hyp-Gly)11 structure.
Three chains in a molecule are shown by different shedding; light-, middle- and dark-gray.
Spheres are water molecules. Intra-chain and inter-chain water-mediated networks are shown in broken and solid lines, respectively.
Threonine in Collagen Triple-helical Structure
Polym. J., Vol. 38, No. 4, 2006 401
carbonyl group of Gly112 in the same chain (broken
lines). In the second case, two water molecules inter-
act between the OH and the amide groups within the
same Thr114 residue (broken lines). And the third
case occurs at two positions; one is two water mole-
cules are linked between the OH group of Thr314
and the carbonyl group of Thr114 of neighboring
chain and another is the similar pattern between the
OH group of Thr214 and the carbonyl group of
Pro317 (solid lines). The average hydrogen bond dis-
tance in these three water-mediated patterns is 2.95 A
The hydration pattern in the second case could not be
found at 4(R)Hyp due to the lack of hydrogen at the
amide group. However, hydration patterns in the first
and the third cases, which are inter- and intra-chain
hydrogen bond networks, are generally observed at
4(R)Hyp in the Yaa position in the peptides including
Pro-4(R)Hyp-Gly tripeptide unit.
These inter- and
intra-chain hydration networks occur repeatedly along
the triple-helical molecule, for example, in (Pro-
4(R)Hyp-Gly)11 structure
as shown in Figure 2b.
They are the dominant feature in the repetitive pat-
terns of 4(R)Hyp in peptides having Pro-4(R)Hyp-
Gly repeating sequence.
Thus, this result indicates
that the OH group of Thr in the Yaa position partici-
pates in water-mediated inter- and intra-chain hydro-
gen bonds in the similar way to the OH group of
4(R)Hyp. Moreover, the position of the OH group
of Thr is located close to that of 4(R)Hyp when
Thr in the OTG structure is superimposed over the
4(R)Hyp in the (Pro-4(R)Hyp-Gly)11 structure (Figure
3). The distance between both hydroxyl oxygen atoms
is about 1 A
˚. The close location of the OH groups of
Thr to 4(R)Hyp could contribute to the similar forma-
tion of water-mediated hydrogen bonds. Therefore,
the OTG structure demonstrates that Thr could act like
4(R)Hyp at OH group side-chain to make similar
water-mediated networks. The thermal stability of Ac-
(Gly-4(R)Hyp-Yaa)10-NH2peptides containing the
Thr is higher than those containing the Ser, alloThr,
Ala and Val,
which suggests the importance of the
side-chain conformation in the triple-helical structure.
So far only the T3-785
peptide has a reported
structure of Thr in a triple-helical structure. The X-
ray determination of the OTG peptide provides insight
into detailed structure of frequently observed residues
in Riftia pachytila cuticle collagen. Although the stabi-
lization mechanism of the OTG peptide is not clearly
understood, the fine structure of the OTG peptide pro-
vides valuable information of Thr conformation in-
cluding diversity of water-mediated hydrogen bonds
around Thr in the triple-helical structure. Interestingly,
the observed hydration patterns of Thr are similar to
those of 4(R)Hyp and moreover, OH group side-chain
characteristic of Thr and 4(R)Hyp is similar as well.
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Figure 3. Superimposition of Thr in the OTG peptide (dark-
gray) on the (Pro-4(R)Hyp-Gly)11 triple-helix (light-gray).
distance between OH groups of Thr and 4(R)Hyp in the Y position
of two peptides is about 1 A
˚and both peptides show the similar
hydration pattern. Gly-4(R)Hyp-Thr in the OTG and Gly-Pro-
4(R)Hyp in the (Pro-4(R)Hyp-Gly)11 are shown in ball and stick
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4(R)Hyp: 4(R)-hydroxyproline
O: 4(R)-hydroxyproline
Gal: galactose
T3-785 peptide: (Pro-Hyp-Gly)3-Ile-Thr-Gly-Ala-
Threonine in Collagen Triple-helical Structure
Polym. J., Vol. 38, No. 4, 2006 403
... We have also observed an increased level of threonine after local cryotherapy exposure in arthritic knee. Threonine is a very important amino acid for the production and stabilization of collagen [49]. It interferes with glycine and serine to strengthen connective tissues and muscles and help them to remain elastic throughout the body [50]. ...
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Rehabilitation using cryotherapy has widely been used in inflammatory diseases to relieve pain and decrease the disease activity. The aim of this study was to explore the metabolite changes in inflammatory knee-joint synovial fluids following local cryotherapy treatment (ice or cold CO 2). We used proton nuclear magnetic resonance (1 H NMR) spectroscopy to assess the metabolite patterns in synovial fluid (SF) in patients with knee arthritis (n = 46) before (D0) and after (D1, 24 h later) two applications of local cryotherapy. Spectra from aqueous samples and organic extracts were obtained with an 11.75 Tesla spectrometer. The metabolite concentrations within the SF were compared between D1 and D0 using multiple comparisons with the application of a false discovery rate (FDR) adjusted at 10% for each metabolite. A total of 32 metabolites/chemical structures were identified including amino acids, organic acids, fatty acids or sugars. Pyruvate, alanine, citrate, threonine was significantly higher at D1 vs D0 (p < 0.05). Tyrosine concentration significantly decreases after cryotherapy application (p < 0.001). We did not observe any effect of gender and cooling technique on metabolite concentrations between D0 and D1 (p > 0.05). The present study provides new insight into a short-term effect of cold stimulus in synovial fluid from patients with knee arthritis. Our observations suggest that the increased level of metabolites involved in energy metabolism may explain the underlying molecular pathways that mediate the antioxidant and anti-inflammatory capacities of cryotherapy.
... These hydroxylations are vital for the structure of collagen, which is comprised uniquely of three left-handed helices wound together around a centralaxis to form a triple stranded right-handed tertiary structure, with multiple collagen molecules cross-linked to form connective tissues. The stability and strength of this structure is dependent upon a Gly-Xaa-Yaa repeating motif, where Xaa is typically L-proline and Yaa is typically 4R-hydroxy-L-proline (Engel and Bächinger, 2005 ). The hydroxylation of proline residues in collagen is catalyzed by procollagen prolyl 3-and 4-hydroxylases (P3H and P4H) to form the 3S-hydroxy-L-proline and 4R-hydroxy-L- proline, respectively (Figure 1A; Myllyharju, 2003; Vranka et al., 2004 ). ...
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Protein hydroxylation has been well-studied in eukaryotic systems. The structural importance of hydroxylation of specific proline and lysine residues during collagen biosynthesis is well established. Recently, key roles for post-translational hydroxylation in signaling and degradation pathways have been discovered. The function of hydroxylation in signaling is highlighted by its role in the hypoxic response of eukaryotic cells, where oxygen dependent hydroxylation of the hypoxia inducible transcription factor both targets it for degradation and blocks its activation. In contrast, the role of protein hydroxylation has been largely understudied in prokaryotes. Recently, an evolutionarily conserved class of ribosomal oxygenases (ROX) that catalyze the hydroxylation of specific residues in the ribosome has been identified in bacteria. ROX activity has been linked to cell growth, and has been found to have a direct impact on bulk protein translation. This discovery of ribosomal protein hydroxylation in bacteria could lead to new therapeutic targets for regulating bacterial growth, as well as, shed light on new prokaryotic hydroxylation signaling pathways. In this review, recent structural and functional studies will be highlighted and discussed, underscoring the regulatory potential of post-translational hydroxylation in bacteria.
... Furthermore, recently reported stabilization effects by Hyp-Hyp-Gly [59] and Hyp-Thr-Gly [60] sequences conflict with our knowledge that the Hyp in the X position destabilizes the triple-helix. Already, several papers have reported studies attempting to understand these matters, based on the structural data at high resolution [37, 38, 51]. However, the work is still very much " in progress. ...
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The triple helix is a specialized protein motif found in all collagens. Although X-ray diffraction studies of collagen began in the 1920s, the very small amount of data available from fiber diffraction of native collagen caused the determination of its molecular conformation to take a very long time. In the early 1950s, two plausible fiber periods of about 20 and 30 A were proposed, together with corresponding single-strand models having 7/2- and 10/3-helical symmetry, respectively. The first framework of the triple helix was proposed by Ramachandran and Kartha in 1955. In the same year, Rich and Crick proposed another structure with the same framework that avoided some of the steric problems of the first model. Their framework, which involved a triple-helical structure with a fiber period of 28.6 A and 10/3-helical symmetry, was exactly the same as one of two single-strand models for collagen proposed at that time, except for the number of strands. At that time, however, nobody considered the triple-strand model with the other framework, with a fiber period of 20 A and 7/2-helical symmetry, until Okuyama et al. detected this structure in the single crystal of (Pro-Pro-Gly)(10) in 1972. Although they proposed this structure as a new structural model for collagen in 1977, it has not been acknowledged as such, but instead has been regarded only as a model for a collagen-like peptide. In 2006, it was shown that both 7/2- and 10/3-helical models could explain X-ray diffraction data from native collagen quantitatively. Furthermore, during the past decade, many single crystals of collagen-model peptides have been analyzed at high resolution. The helical symmetries observed in these model peptides are very close to the ideal 7/2-helical symmetry, whereas no supporting data were found for the 10/3-helical model. This evidence strongly suggests that an average molecular structure of native collagen is the 7/2-helical model rather than the prevailing Rich and Crick (10/3-helical) model. Knowing the correct molecular structure, the driving force for the formation of a quarter-staggered structure in collagen fibrils will be elucidated in the near future by analysis incorporating the molecular structure of collagen and its amino acid sequence.
Click chemistry was used to introduce moieties as sterically demanding as monosaccharides into the Yaa position of collagen model peptides. The effect of different triazolyl derivatives as well as the configuration of the functionalized proline residue on the thermal stability of the collagen triple helices was examined.
The single-crystal structures of three collagen-like host-guest peptides, (Pro-Pro-Gly)(4) -Hyp-Yaa-Gly-(Pro-Pro-Gly)(4) [Yaa = Thr, Val, Ser; Hyp = (4R)-4-hydroxyproline] were analyzed at atomic resolution. These peptides adopted a 7/2-helical structure similar to that of the (Pro-Pro-Gly)(9) peptide. The stability of these triple helices showed a similar tendency to that observed in Ac-(Gly-Hyp-Yaa)(10) -NH(2) (Yaa = Thr, Val, Ser) peptides. On the basis of their detailed structures, the differences in the triple-helical stabilities of the peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence were explained in terms of van der Waals interactions and dipole-dipole interaction between the Hyp residue in the X position and the Yaa residue in the Y position involved in the Hyp(X):Yaa(Y) stacking pair. This idea also explains the inability of Ac-(Gly-Hyp-alloThr)(10) -NH(2) and Ac-(Gly-Hyp-Ala)(10) -NH(2) peptides to form triple helices. In the Hyp(X):Thr(Y), Hyp(X):Val(Y), and Hyp(X):Ser(Y) stacking pairs, the proline ring of the Hyp residues adopts an up-puckering conformation, in agreement with the residual preference of Hyp, but in disagreement with the positional preference of X in the Gly-Xaa-Yaa sequence.
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The average crystal structure of a collagen-model peptide, (Pro-Pro-Gly)9 has been determined at 1.0Å resolution. Crystals belong to an orthorhombic system (P212121) with cell parameters of a=26.82(2), b=26.33(2), and c=20.25(2)Å. The X-Ray oscillation photograph clearly showed satellite spots with an 80 Å axial repeat on both sides of the strong spots on the layer lines corresponding to c=20Å. The existence of c=80Å axial repeat suggests that the longer repeat along the c-axis is the shortest integral multiply of the helical repeat (20Å) enough to accommodate one (Pro-Pro-Gly)9 molecule and an appropriate gap between adjacent molecules. According to the reflection data with c=20Å axial repeat, the overall peptide structure is very close to the left-handed 7/2-helical model for collagen. Based on the difference Fourier map, 34 water molecules were added in the asymmetric unit of seven triplets. Of which 14 water molecules make hydrogen bonds with peptide chains and rest of them participate in hydrogen bonds only with other water molecules.
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The 2 A crystal structure reported here of the collagen-like model peptide, T3-785, provides the first visualization of how the sequence of collagen defines distinctive local conformational variations in triple-helical structure.
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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.
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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.
Two types of annelid collagens of different sizes were purified, one from acetic acid extracts of the cuticle (length 2.5 microns) and the other, after pepsin digestion, from interstitial spaces of the body wall (0.3 micron). They were obtained from Alvinella pompejana, Alvinella caudata and Paralvinella grasslei collected at 2600 m depth around anoxic hydrothermal vents and from Arenicola marina and Nereis diversicolor living in shallow sea-water habitats. The length of the corresponding collagens from different species and their amino acid compositions including the hydroxylation of proline were remarkably similar. The melting point of the triple helix, however, differed between the Alvinella species (approximately 45 degrees C), Paralvinella (approximately 35 degrees C) and the shallow sea-water annelids (approximately 28 degrees C), indicating adaption to habitats with different temperatures. The cuticle collagens of the annelids possess a globular domain, which is apparently involved in oligomer formation, and show similar fragment pattern. Almost identical cross-striation patterns of segment-long-spacing segments of the interstitial collagens indicated sequence similarity, which was confirmed by partial Edman degradation of alpha-chains. These data showed almost complete identity between the two Alvinella species and a lower sequence identity with Paralvinella (approximately 95%), Arenicola (67 to 72%) and the vent vestimentiferan Riftia pachyptila (64 to 71%). The data suggest a close evolutionary relationship between these worms, despite a clear separation of habitat preference and thermal stability of the collagens.
Invertebrates comprise about 95% of animal species, yet most studies of extracellular matrices have centered on vertebrates. Comparative studies of invertebrates will enhance comprehension of evolutionary processes and appreciation of the diversity of extracellular matrices. Moreover, new functions and new structures will be revealed over a wide range of organismic needs. Another important perspective is that several invertebrate species have provided insight into developmental processes, and those processes often have direct relevance to vertebrate development. Thus, studies of fruit flies, nematodes, and sea urchins have revealed common features of cell biology, embryonic development, and matrix properties that pertain throughout the animal kingdom. The advantages of invertebrates are their rapid rates of embryonic development, their amenability to genetic manipulation, availability of innumerable mutants, and their ease of study in the laboratory. Extracellular matrices themselves are readily compared. Invertebrates display a wide diversity of such matrices, at the levels of both tissue architecture and molecular anatomy. Knowledge of that diversity leads to an appreciation of evolutionary variety and eventually to comprehension of the organization of extracellular matrices and of the properties of their constituent macromolecules. The expanding knowledge of unique matrix molecules from invertebrates also has economic potential and is beginning to provide new materials for biotechnology.
The collagen triple helix is a unique protein motif defined by the supercoiling of three polypeptide chains in a polyproline II conformation. It is a major domain of all collagen proteins and is also reported to exist in proteins with host defense function and in several membrane proteins. The triple-helical domain has distinctive properties. Collagen requires a high proportion of the post-translationally modified imino acid 4-hydroxyproline and water to stabilize its conformation and assembly. The crystal structure of a collagen-like peptide determined to 1.85 Angstrum showed that these two features may be related. A detailed analysis of the hydration structure of the collagen-like peptide is presented. The water molecules around the carbonyl and hydroxyprolyl groups show distinctive geometries. There are repetitive patterns of water bridges that link oxygen atoms within a single peptide chain, between different chains and between different triple helices. Overall, the water molecules are organized in a semi-clathrate-like structure that surrounds and interconnects triple helices in the crystal lattice. Hydroxyprolyl groups play a crucial role in the assembly. The roles of hydroxyproline and hydration are strongly interrelated in the structure of the collagen triple helix. The specific, repetitive water bridges observed in this structure buttress the triple-helical conformation. The extensively ordered hydration structure offers a good model for the interpretation of the experimental results on collagen stability and assembly.
The cuticle collagen of the vestimentiferan Riftia pachyptila, an organism which is endemic to deep-sea hydrothermal vents, has several unusual properties including an extraordinary length (1.5 microns), a high thermal stability (37 degrees C) in spite of a low 4-hydroxyproline content and an atypically high threonine content (20 mol%). We have now purified the constituent chain of cuticle collagen and show that it contains about 40% carbohydrate, which is mainly galactose, indicating that the chain has a molecular mass of approximately 750 kDa. Several large (30 to 150 kDa) fragments, which all contained carbohydrate, could be produced by cleavage with endoproteinase Lys-C, bacterial collagenase and cyanogen bromide (CNBr). Edman degradation of these and several smaller fragments was used to determine about 3000 sequence positions comprising 60% of the total triple-helical sequence. This demonstrated mainly typical Gly-X-Y triplet repeats with a few imperfections and a longer N-terminal non-triplet sequence. Most of the 4-hydroxyproline was found in triplet position X, where it decreases the stability of the triple helix. About 40% of the Y positions could not be identified, which correlated with a low abundance of threonine in the sequence and the demonstration of threonine in these positions after deglycosylation of several peptides by treatment with hydrofluoric acid. Matrix-assisted laser desorption ionisation mass spectrometry of selected peptides indicated that the blocked threonine residues are occupied by chains of one, two or three hexoses (presumably galactose). These glycosylated threonine residues in Y positions are therefore likely to replace 4-hydroxyproline as the major contributor to triple helix stabilization. Studies with a synthetic (Gly-Pro-Thr)10 oligopeptide demonstrated a low thermal stability of its triple helix which emphasizes a crucial role of glycosylation for stabilization.
For most collagens, the melting temperature (T(m)) of the triple-helical structure of collagen correlates with the total content of proline (Pro) and 4-trans-hydroxyproline (Hyp) in the Xaa and Yaa positions of the -Gly-Xaa-Yaa- triplet repeat. The cuticle collagen of the deep-sea hydrothermal vent worm Riftia pachyptila, despite a very low content of Pro and Hyp, has a relatively high thermal stability. Rather than Hyp occupying the Yaa position, as is normally found in mammalian collagens, this position is occupied by threonine (Thr) which is O-glycosylated. We compare the triple-helix forming propensities in water of two model peptides, Ac-(Gly-Pro-Thr)(10)-NH(2) and Ac-(Gly-Pro-Thr(Galbeta))(10)-NH(2), and show that a collagen triple-helix structure is only achieved after glycosylation of Thr. Thus, we show for the first time that glycosylation is required for the formation of a stable tertiary structure and that this modification represents an alternative way of stabilizing the collagen triple-helix that is independent of the presence of Hyp.
The glycopeptide Ac-(Gly-Pro-Thr(beta-Gal))(10)-NH(2) forms a collagen-like triple-helix. A (1)H NMR structural analysis is reported for the peptides Ac-(Gly-Pro-Thr)(n)-NH(2) and Ac-(Gly-Pro-Thr(beta-Gal))(n)-NH(2), where n = 1, 5, and 10. NMR assignments for the individual peptides are made using one- and two-dimensional TOCSY, ROESY, and NOESY experiments. The NMR and corroborating CD data show that Ac-(Gly-Pro-Thr)(n)-NH(2), n = 1, 5, or 10, as well as Ac-(Gly-Pro-Thr(beta-Gal))(n)-NH(2), n = 1 or 5 peptides are unable to form collagen-like triple-helical structures. Furthermore, the equilibrium ratio of cis to trans isomers of the Pro residues is unaffected by the presence of carbohydrate. For Ac-(Gly-Pro-Thr(beta-Gal))(10)-NH(2), the kinetics of amide (1)H exchange with solvent deuterium indicate a slow rate of exchange for both the Gly and the Thr amide. The data are thus consistent with a model in which the carbohydrate stabilizes the triple helix through an occlusion of water molecules and by hydrogen bonding but not through an influence on the cis to trans isomer ratio.