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PDBsum entry 2z30
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References listed in PDB file
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Key reference
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Title
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Four new crystal structures of tk-Subtilisin in unautoprocessed, Autoprocessed and mature forms: insight into structural changes during maturation.
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Authors
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S.Tanaka,
H.Matsumura,
Y.Koga,
K.Takano,
S.Kanaya.
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Ref.
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J Mol Biol, 2007,
372,
1055-1069.
[DOI no: ]
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PubMed id
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Abstract
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Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis
(Tk-subtilisin) is matured from Pro-Tk-subtilisin upon autoprocessing and
degradation of the propeptide. The crystal structures of the autoprocessed and
mature forms of Tk-subtilisin were determined at 1.89 A and 1.70 A resolution,
respectively. Comparison of these structures with that of unautoprocessed
Pro-Tk-subtilisin indicates that the structure of Tk-subtilisin is not seriously
changed during maturation. However, one unique Ca(2+)-binding site (Ca-7) is
identified in these structures. In addition, the N-terminal region of the mature
domain (Gly70-Pro82), which binds tightly to the main body in the
unautoprocessed form, is disordered and mostly truncated in the autoprocessed
and mature forms, respectively. Interestingly, this site is formed also in the
unautoprocessed form when its crystals are soaked with 10 mM CaCl(2), as
revealed by the 1.87 A structure. Along with the formation of this site, the
N-terminal region (Leu75-Thr80) is disordered, with the scissile peptide bond
contacting with the active site. These results indicate that the calcium ion
binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon
autoprocessing. We propose that the Ca-7 site is required to promote the
autoprocessing reaction by stabilizing the autoprocessed form, in which the new
N terminus of the mature domain is structurally disordered. Furthermore, the
crystal structure of the Tk-propeptide:S324A-subtilisin complex, which was
formed by the addition of separately expressed proteins, was determined at 1.65
A resolution. This structure is virtually identical with that of the
autoprocessed form, indicating that the interaction between the two domains is
highly intensive and specific.
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Figure 5.
Figure 5. The seventh Ca^2+-binding site. (a) A stereo view
of electron density around the Ca-7 site of the mature form is
shown. The 2F[o]–F[c] map contoured at the 1.5σ and 5.0σ
levels are shown in blue and magenta, respectively. The calcium
ion and the water molecules are represented by cyan and red
spheres, respectively. The residues and water molecules that
coordinate with the calcium ion are labeled. (b) The structures
of the Ca-7 sites of the autoprocessed form (lime green), mature
form (orange), and unautoprocessed form 2 (gray) are
superimposed. The residues that coordinate with the calcium ion
are shown as a stick model.
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Figure 6.
Figure 6. Stereo view of the N-terminal region of the mature
domain. The structures of the autoprocessed form (lime green),
mature form (orange), and unautoprocessed form 2 (gray) shown as
a stick model are superimposed. Two hydrogen bonds between Gln81
O^ε1 and Tyr317 O^γ, and between Gln81 N^ε2 and Asp315 O, are
shown with broken lines.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
372,
1055-1069)
copyright 2007.
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