Threonine in Collagen Triple-helical Structure
Nattha JIRAVANICHANUN,1;2Kazunori MIZUNO,3Hans Peter BA
¨CHINGER,3and Kenji OKUYAMA1;
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 /
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: firstname.lastname@example.org).
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.
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-
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-
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
Thr314 O 1γ
Thr114 N Hyp4B Oδ
Thr114 O 1
Thr114 O Thr214 O 1
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-
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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T3-785 peptide: (Pro-Hyp-Gly)3-Ile-Thr-Gly-Ala-
Threonine in Collagen Triple-helical Structure
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