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Leikin demonstrate that the main obstacle to achieving
the native triple-helical conformation is not aggregation
or misfolding of procollagen chains but rather the intrin-
sic thermal instability of the native, folded state. In phos-
phate-buffered saline as well as in buffers that mimic
the protein concentration and ionic environment of ER,
human procollagen triple helix spontaneously folds into
its native conformation at 30-34°C but not at higher tem-
peratures. To fold at body temperature, the triple-helix
conformation has to be stabilized through interactions
with chaperone molecules that preferentially bind to the
native triple helix. Based on the triple-helix folding tem-
peratures and published binding constants, Marakeeva
and Leikin concluded that HSP47 is likely to perform
this function. They also report that it takes more than 20
HSP47 molecules to stabilize a single triple helix at body
temperature.
19
which corresponded to the reactive center in serpin
family proteins. Mutagenic studies suggested that tryp-
tophan (Trp) 280 is involved in the interaction with col-
lagen based on the observation that the binding affinity
to collagen was significantly compromised by an alanine
substitution of Trp-280. Furthermore, these investigators
probed the collagen binding site of HSP47 and observed
chemical shift perturbations (His-261 and His-373) and/
or peak intensity attenuations (His-202 and His-225)
upon addition of the trimeric collagen peptide. In addi-
tion, the mutational analysis indicates that the amino
acid substitutions in the B/C β-barrel domain and the
loop (M223A, Y230F, W280A and M363A) significantly
compromised the interaction with collagen, indicat-
ing that Met223, Met363 and Tyr230 are involved in the
HSP47/collagen interaction.
From the standpoint of affinity sites on the collagen
triple helix, Koide et al. examined HSP47 binding to the
collagen triple helix.
14
Various collagen synthetic pep-
tides were synthesized and their interaction with HSP47
in vitro
was examined. These investigators found that
the Pro-Arg-Gly triplet as found in the type I collagen
N-terminal domain forms an HSP47-binding site. Using
model peptides, HSP47 binding was observed only when
Arg residues were incorporated in the Yaa positions of
the Xaa-Yaa-Gly triplets. Amino acids in the Xaa position
did not largely affect the interaction. The recognition of
the Arg residue by HSP47 was specific to its side-chain
structure because replacement of the Arg residue by other
basic amino acids decreased the affinity to HSP47.
14
The amino acid at the Yaa position (Yaa-3) in the
N-terminal-adjoining triplet containing the critical Arg
(defined as Arg(0)) was also considered as directly recog-
nized by HSP47.
15
Based on these findings, the relation-
ship between the structure of Yaa(-3) and HSP47 binding
was examined using synthetic collagenous peptides. The
results indicated that the structure of Yaa(-3) determined
the binding affinity for HSP47. Moreover, the required
relative spatial arrangement of these key residues in the
triple helix was analyzed by taking advantage of hetero-
trimeric collagen-model peptides, each of which contains
one Thr(-3) and one Arg(0). The results revealed that
HSP47 recognizes the Yaa(-3) and Arg(0) residues only
when they are on the same peptide strand.
15
In related studies, Nishikawa et al. demonstrated
that HSP47 detects the side-chain structure of Arg at
the Yaa(0) position, while the Yaa(-3) amino acid serves
as the secondary recognition site that affects affinity to
HSP47.
24
Widmer et al. studied HSP47/collagen interac-
tion using the crystal structure of recombinant canine
HSP47 in free form and in complex with three homotri-
meric synthetic collagen model peptides. It is important
to point out that the other studies reviewed here used
HSP47 from different species.
HSP47 BINDING TO THE COLLAGEN
TRIPLE HELIX
Several studies summarized below have recently
addressed the mechanisms involved in binding of HSP47
to the collagen triple helix as well as the affinity sites for
HSP47 on the collagen molecule. In general, these stud-
ies have analyzed binding sites on model peptides. The
critical issues are: (1) where HSP47 binds to the collagen
triple helix and (2) how it is released in the Golgi and
recycled to the ER after procollagen processing has been
completed.
Noting that in the ER HSP47 binds procollagen at
a neutral pH but in the Golgi releases it at an acidic pH
similar to the p
K
(a) of the imidazole side chain of histi-
dine residues, Abdul-Wahab et al. examined three clus-
ters of the 14 histidine residues on murine HSP47, termed
the “breach, gate and shutter” groups.
23
Employing histi-
dine mutagenesis, the study demonstrated that His-198,
His-197 and His-191 were important (if not central) to the
HSP47 mechanism of binding/release to collagen. The
suggestion that these histidine residues were central to
the “pH switch” was consistent with the breach cluster
residues being well conserved across the HSP47 family.
To examine HSP47 binding to collagen Yagi-Utsumi
et al. performed nuclear magnetic resonance (NMR)
spectroscopic analyses to characterize the interaction
in solution between chicken HSP47 and a synthetic col-
lagen peptide trimer.
9
Selective (15)N-labeling was
used to target tryptophan and histidine residues. Site-
directed mutagenesis of the individual residues was then
employed to assess spectral changes observed upon the
interaction of HSP47 with the collagen peptide. Using
a three-dimensional-homology model of HSP47, these
investigators mapped the collagen-binding site to the
B/C β-barrel domain and a spatially proximal loop
