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PDBsum entry 3b45
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Membrane protein
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PDB id
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3b45
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References listed in PDB file
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Key reference
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Title
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The role of l1 loop in the mechanism of rhomboid intramembrane protease glpg.
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Authors
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Y.Wang,
S.Maegawa,
Y.Akiyama,
Y.Ha.
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Ref.
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J Mol Biol, 2007,
374,
1104-1113.
[DOI no: ]
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PubMed id
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Abstract
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Intramembrane proteases are important enzymes in biology. The recently solved
crystal structures of rhomboid protease GlpG have provided useful insights into
the mechanism of these membrane proteins. Besides revealing an internal
water-filled cavity that harbored the Ser-His catalytic dyad, the crystal
structure identified a novel structural domain (L1 loop) that lies on the side
of the transmembrane helices. Here, using site-directed mutagenesis, we
confirmed that the L1 loop is partially embedded in the membrane, and showed
that alanine substitution of a highly preferred tryptophan (Trp136) at the
distal tip of the L1 loop near the lipid:water interface reduced GlpG
proteolytic activity. Crystallographic analysis showed that W136A mutation did
not modify the structure of the protease. Instead, the polarity for a small and
lipid-exposed protein surface at the site of the mutation has changed. The
crystal structure, now refined at 1.7 A resolution, also clearly defined a
20-A-wide hydrophobic belt around the protease, which likely corresponded to the
thickness of the compressed membrane bilayer around the protein. This improved
structural model predicts that all critical elements of the catalysis, including
the catalytic serine and the L5 cap, need to be positioned within a few
angstroms of the membrane surface, and may explain why the protease activity is
sensitive to changes in the protein:lipid interaction. Based on these findings,
we propose a model where the end of the substrate transmembrane helix first
partitions out of the hydrophobic core region of the membrane before it bends
into the protease active site for cleavage.
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Figure 4.
Fig. 4. The crystal structure of W136A mutant. (a) Electron
density, contoured at 1.5σ level, at the site of mutation.
Water molecules are shown in red, and detergents in green. (b)
The water molecules (numbered 1 to 6) substituting the indole
ring of Trp136 are stabilized by a network of hydrogen bonds.
(c) The structure of the wild type showing the interfacial
location of Trp136. (d) After mutation, the protein surface is
no longer compatible with its buried location.
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Figure 5.
Fig. 5. The model of membrane-embedded GlpG. (a) The C^α
trace of the protein is shown in yellow, externally bound water
in red, and water inside the membrane protein in blue. The water
molecules substituting for Trp136 in the mutant are shown in
white. The two horizontal lines mark the boundaries of the
hydrophobic core of the membrane around the protease. (b) A
histogram of the number of water molecules observed across the
bilayer in the same color scheme as in (a). (c) The location of
various structural elements important for protease function
within the lipid bilayer. Parts of L1 (marked by *) and TM helix
S2 (**) are omitted to show the internal active site.
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The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
374,
1104-1113)
copyright 2007.
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