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Threonine in Collagen Triple-helical Structure

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Abstract

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.
SHORT COMMUNICATIONS
Threonine in Collagen Triple-helical Structure
Nattha JIRAVANICHANUN,1;2Kazunori MIZUNO,3Hans Peter BA
¨CHINGER,3and Kenji OKUYAMA1;
y
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.
1,2
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-
cies.
3,4
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.
5
The mechanism of the stability of the collagen helix
is still unknown. The Thr of the collagen is highly gly-
cosylated.
4
Several model peptides were synthesized
to analyze its thermal stability and property.
5–9
The
O-galactosylation of Thr increases the thermal stability
(the helix-coil transition temperature) of Ac-(Gly-Pro/
4(R)Hyp-Thr)10-NH2peptides.
6
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.
9
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
method.
9
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,
6
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-
Gly)9.
10
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.
EXPERIMENTAL
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
CrystalClear.
11
Crystal belongs to monoclinic space
y
To whom correspondence should be addressed (Tel: +81-66-850-5455, Fax: +81-66-850-5455, E-mail: okuyamak@chem.sci.osaka-u.ac.jp).
400
Polymer Journal, Vol. 38, No. 4, pp. 400–403 (2006)
group P21with unit cell parameters a¼26:0,b¼
26:5,c¼80:2A
˚,¼90:4. The structure of OTG
was determined by molecular replacement method
using (Pro-Pro-Gly)9peptide (PDB code 1ITT)
12
as
a search model. Positional refinement was performed
by X-PLOR
13
and structure refinement was carried
out by SHELX-L.
14
RESULTS AND DISCUSSION
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-
tide.
15
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.
10,16
In T3-785 structure,
15
water-
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
gauche+
N
Oγ1
tran
s
Cγ2
Figure 1. gaucheþ/trans conformation of Thr in the OTG
structure.
Gly1A O
Gly2B O
Gly112 O
Thr314 O 1γ
Hyp3A O
Thr114 N Hyp4B Oδ
δ
δ
Thr114 O 1
Hyp5C O
Hyp4B O
Thr114 O Thr214 O 1
γ
γ
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.
16
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.
16,17
These inter- and
intra-chain hydration networks occur repeatedly along
the triple-helical molecule, for example, in (Pro-
4(R)Hyp-Gly)11 structure
16
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.
16,17
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,
9
which suggests the importance of the
side-chain conformation in the triple-helical structure.
So far only the T3-785
15
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.
REFERENCES
1. J. Myllyharju and K. I. Kivirikko, Trends Genet.,20,33
(2004).
2. C. M. Kielty and M. E. Grant, ‘‘Connective tissue and
its heritable disorders. Molecular Genetics in Medical As-
pects,’’ 2nd ed., Wiley Liss, New York, 2002, pp 159–221.
3. R. Har-El and M. L. Tanzer, FASEB J.,7, 1115 (1993).
4. F. Gaill, K. Mann, H. Wiedemann, J. Engel, and R. Timpl,
J. Mol. Biol.,246, 284 (1995).
5. K. Mann, D. E. Mechling, H. P. Ba
¨chinger, C. Eckerskorn,
F. Gaill, and R. Timpl, J. Mol. Biol.,261, 255 (1996).
6. J. G. Bann and H. P. Ba
¨chinger, J. Biol. Chem.,275, 24466
(2000).
7. J. G. Bann, D. H. Peyton, and H. P. Ba
¨chinger, FEBS Lett.,
473, 237 (2000).
8. J. G. Bann, H. P. Ba
¨chinger, and D. H. Peyton, Biochemis-
try,42, 4042 (2003).
9. K. Mizuno, T. Hayashi, and H. P. Ba
¨chinger, J. Biol. Chem.,
278, 32373 (2003).
10. C. Hongo, K. Noguchi, K. Okuyama, Y. Tanaka, and N.
Nishino, J. Biochem.,138, 135 (2005).
11. CrystalClear (Rigaku) Molecular Structure Corporation, The
Woodlands, Texas, USA, 1999.
12. C. Hongo, V. Nagarajan, K. Noguchi, S. Kamitori, K.
Okuyama, Y. Tanaka, and N. Nishino, Polym. J.,33, 812
(2001).
13. A. T. Brunger, ‘‘X-PLOR Version 3.1 System for X-ray
Crystallography and NMR,’’ Yale University Press, New
Haven: CT, 1992.
1 Å
Figure 3. Superimposition of Thr in the OTG peptide (dark-
gray) on the (Pro-4(R)Hyp-Gly)11 triple-helix (light-gray).
16
The
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
diagram.
N. JIRAVANICHANUN et al.
402 Polym. J., Vol. 38, No. 4, 2006
14. G. M. Sheldric and T. R. Schneidern, Methods Enzymol.,
277, 319 (1997).
15. R. Z. Kramer, J. Bella, P. Mayville, B. Brodsky, and H. M.
Berman, Nat. Struct. Biol.,6, 454 (1999).
16. K. Okuyama, C. Hongo, R. Fukushima, G. Wu, H. Narita, K.
Noguchi, Y. Tanaka, and N. Nishino, Biopolymers,76, 367
(2004).
17. J. Bella, B. Brodsky, and H. M. Berman, Structure,3, 893
(1995).
4(R)Hyp: 4(R)-hydroxyproline
O: 4(R)-hydroxyproline
Gal: galactose
T3-785 peptide: (Pro-Hyp-Gly)3-Ile-Thr-Gly-Ala-
Arg-Gly-Leu-Ala-Gly-Pro-Hyp-Gly-(Pro-Hyp-Gly)3
Threonine in Collagen Triple-helical Structure
Polym. J., Vol. 38, No. 4, 2006 403
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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.
Article
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.
Article
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.
Article
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.