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PDBsum entry 2e1p
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References listed in PDB file
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Key reference
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Title
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Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for ca2+-Induced folding.
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Authors
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S.Tanaka,
K.Saito,
H.Chon,
H.Matsumura,
Y.Koga,
K.Takano,
S.Kanaya.
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Ref.
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J Biol Chem, 2007,
282,
8246-8255.
[DOI no: ]
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PubMed id
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Abstract
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The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A)
from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at
2.3 A resolution. The overall structure of this protein is similar to those of
bacterial subtilisin-propeptide complexes, except that the peptide bond linking
the propeptide and mature domain contacts with the active site, and the mature
domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial
subtilisins but is formed prior to autoprocessing, unlike the corresponding
sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the
pro-S324A structure and are located at the surface loops. Four of them
apparently contribute to the stability of the central alphabetaalpha
substructure of the mature domain. The CD spectra,
1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to
chymotryptic digestion of this protein indicate that the conformation of
pro-S324A is changed from an unstable molten globule-like structure to a stable
native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown
to be autoprocessed to form a propeptide-mature domain complex in the presence
of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C
is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon
subsequent autoprocessing. These results suggest that the maturation process of
Tk-subtilisin is different from that of bacterial subtilisins in terms of the
requirement of Ca2+ for folding of the mature domain and completion of the
folding process prior to autoprocessing.
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Figure 2.
FIGURE 2. A stereo view of electron density around the
active site (A) and Ca-1 site (B) of pro-S324A. In A, the 2F[o]
- F[c] map contoured at the 1.5  level is shown. In B,
the 2F[o] - F[c] maps contoured at the 2.0 and 5.0 levels
are shown in blue and magenta, respectively. The active site
residues, the nitrogen and oxygen atoms, and the Ca^2+ ions are
indicated as in Fig. 1.
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Figure 3.
FIGURE 3. Ca^2+ binding sites. The structures of the Ca-1
(A), Ca-2 to Ca-5 (B), and Ca-6 (D) sites are shown. The Ca^2+
ions and water molecules are represented by spheres. The
residues that bind to the Ca^2+ ions are labeled. In C, a stereo
view of the central   substructure consisting
of the 6m-helix, 5m-strand, and 7m-helix
and the 1m-strand are shown in a
ribbon drawing. One of the active site residues, Asp^115, is
indicated by a stick model.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
8246-8255)
copyright 2007.
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