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PDBsum entry 2z56
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References listed in PDB file
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Key reference
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Title
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Requirement of left-Handed glycine residue for high stability of the tk-Subtilisin propeptide as revealed by mutational and crystallographic analyses.
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Authors
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M.A.Pulido,
S.Tanaka,
C.Sringiew,
D.J.You,
H.Matsumura,
Y.Koga,
K.Takano,
S.Kanaya.
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Ref.
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J Mol Biol, 2007,
374,
1359-1373.
[DOI no: ]
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PubMed id
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Abstract
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Tk-subtilisin [the mature domain of Pro-Tk-subtilisin in active form
(Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis
is matured from Pro-Tk-subtilisin [a subtilisin homologue from T. kodakaraensis
in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide.
Pro-Tk-subtilisin is characterized by extremely slow maturation at mild
temperatures, but this maturation rate is greatly increased by a single
Gly56-->Ser mutation in the propeptide region. To analyze the role of Gly56,
which assumes a left-handed conformation, Pro-Tk-subtilisin variants with
complete amino acid substitutions at Gly56 were constructed. A comparison of
their halo-forming activities suggests that all variants, except for Pro-G56W
[Pro-G56X, Pro-Tk-subtilisin with Gly56-->X mutation (X = any amino acid)],
mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest
maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S,
were overproduced, purified, and characterized. SDS-PAGE analyses and
Tk-subtilisin activity assay indicated that their maturation rates increased in
the order WT < or = Pro-G56W < Pro-G56S < Pro-G56E. The propeptides of
these variants were also overproduced, purified, and characterized. The
stability and inhibitory potency of these propeptides decreased in the order
Tk-propeptide [propeptide of Tk-subtilisin (Gly1-Leu69)] > or = G56W-propeptide
> G56S-propeptide > G56E-propeptide, indicating that they are inversely
correlated with the maturation rates of Pro7-Tk-subtilisin and its derivatives.
The crystal structures of these propeptides determined in complex with
S324A-subtilisin indicate that the conformation of the propeptide is altered by
the mutation, such that nonglycine residues at position 56 assume a right-handed
conformation and hydrophobic interactions at the core region decrease. These
results indicate that Gly56 is required in stabilizing the propeptide fold.
Stabilization of this fold leads to strong binding of Tk-propeptide to
Tk-subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and
slow maturation of Pro-Tk-subtilisin.
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Figure 1.
Fig. 1. Halo-forming activities of the wild-type and mutant
proteins of Pro-Tk-subtilisin. The halo-forming activities of
Pro-Tk-subtilisin (WT), Pro-G56S, Pro-G56W, and Pro-G56E were
analyzed at the indicated temperatures as described in Materials
and Methods. The E. coli HB101 transformant with pBR322 serves
as a negative control.
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Figure 7.
Fig. 7. Stereo views of the three-dimensional structures of
the propeptide:subtilisin complexes. (a) The entire structure of
the G56S-propeptide:S324A-subtilisin complex is superimposed on
that of the Tk-propeptide:S324A-subtilisin complex (Protein Data
Bank code 2Z30). For the structure of the
G56S-propeptide:S324A-subtilisin complex, G56S-propeptide is in
red and S324A-subtilisin is in green. Two active-site residues
(Asp115 and His153) and Ala324, which is substituted for the
active-site serine residue, are indicated by yellow stick
models, in which the oxygen and nitrogen atoms are in red and
blue, respectively. Seven Ca^2 + are shown in cyan spheres. N
and C represent the N- and C-termini, respectively. The entire
structure of the Tk-propeptide:S324A-subtilisin complex,
including seven Ca^2 +, is in gray. (b–d) G56S-propeptide (b),
G56E-propeptide (c), and G56W-propeptide (d) in the structures
of the G56S-propeptide:S324A-subtilisin,
G56E-propeptide:S324A-subtilisin, and
G56W-propeptide:S324A-subtilisin complexes are superimposed on
Tk-propeptide in the structure of the
Tk-propeptide:S324A-subtilisin complex. The view direction is
changed from (a), such that the mature domain is located behind
the propeptide. The mutant and wild-type propeptides are in red
and gray, respectively. Nine of the 11 residues that form a
hydrophobic core and Ser56 (b), Glu56 (c), or Trp56 (d) are
indicated by blue and yellow stick models, respectively. Two
other core-forming residues are not shown because they are
hidden.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
374,
1359-1373)
copyright 2007.
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