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PDBsum entry 2in8
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References listed in PDB file
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Key reference
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Title
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Crystallographic and mutational studies of mycobacterium tuberculosis reca mini-Inteins suggest a pivotal role for a highly conserved aspartate residue.
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Authors
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P.Van roey,
B.Pereira,
Z.Li,
K.Hiraga,
M.Belfort,
V.Derbyshire.
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Ref.
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J Mol Biol, 2007,
367,
162-173.
[DOI no: ]
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PubMed id
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Abstract
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The 440 amino acid Mtu recA intein consists of independent protein-splicing and
endonuclease domains. Previously, removal of the central endonuclease domain of
the intein, and selection for function, generated a 168 residue mini-intein,
DeltaI-SM, that had splicing activity similar to that of the full-length,
wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage
activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide,
we previously generated a highly active 139 residue mini-intein,
DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual
endonuclease loop with a seven-residue beta-turn from the autoprocessing domain
of Hedgehog protein. The three-dimensional structures of DeltaI-SM,
DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh),
have been determined to evaluate the effects of the minimization on intein
integrity and to investigate the structural and functional consequences of the
D422G mutation. These structural studies show that Asp422 is capable of
interacting with both the N and C termini. These interactions are lacking in the
CM variant, but are replaced by contacts with water molecules. Accordingly,
additional mutagenesis of residue 422, combined with mutations that isolate
N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a
role in both N and C-terminal cleavage, thereby suggesting that this highly
conserved residue regulates the balance between the two reactions.
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Figure 2.
Figure 2. Structure of ΔI-SM. (a) Molecular structure of
ΔI-SM. (b) Comparison of the conformation of ΔI-SM (green)
with the GyrA intein^12 (red). (c) Dimer observed in the
asymmetric unit, with zinc ions shown in cyan. (d) Coordination
of the zinc ion by residues Glu424, His429, and the C-terminal
aminosucciminide (SUC440) from one molecule (green), and His439
from the second molecule (red) in the asymmetric unit. Figure
2. Structure of ΔI-SM. (a) Molecular structure of ΔI-SM. (b)
Comparison of the conformation of ΔI-SM (green) with the GyrA
intein[3]^12 (red). (c) Dimer observed in the asymmetric unit,
with zinc ions shown in cyan. (d) Coordination of the zinc ion
by residues Glu424, His429, and the C-terminal aminosucciminide
(SUC440) from one molecule (green), and His439 from the second
molecule (red) in the asymmetric unit.
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Figure 4.
Figure 4. Effect of the D422G mutation. (a) Superposition of
the structures of ΔΔI[hh]-SM (green) and ΔΔI[hh]-CM (red).
(b) Detailed comparison of the area of residue 422. Water
molecules in this area are shown as spheres, light green for
ΔΔI[hh]-SM and rose for ΔΔI[hh]-CM. In ΔΔI[hh]-CM, two
water molecules are found bound tightly to the protein in the
area of the missing Asp422 side-chain. Figure 4. Effect of
the D422G mutation. (a) Superposition of the structures of
ΔΔI[hh]-SM (green) and ΔΔI[hh]-CM (red). (b) Detailed
comparison of the area of residue 422. Water molecules in this
area are shown as spheres, light green for ΔΔI[hh]-SM and rose
for ΔΔI[hh]-CM. In ΔΔI[hh]-CM, two water molecules are found
bound tightly to the protein in the area of the missing Asp422
side-chain.
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The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
367,
162-173)
copyright 2007.
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