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PDBsum entry 2e3h
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Structural protein
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PDB id
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2e3h
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References listed in PDB file
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Key reference
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Title
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Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.
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Authors
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M.Mishima,
R.Maesaki,
M.Kasa,
T.Watanabe,
M.Fukata,
K.Kaibuchi,
T.Hakoshima.
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Ref.
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Proc Natl Acad Sci U S A, 2007,
104,
10346-10351.
[DOI no: ]
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PubMed id
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Abstract
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Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus
end-tracking proteins that regulate microtubule dynamics, but it is obscure how
CLIP-170 recognizes the microtubule plus end and contributes to polymerization
rescue. Crystallographic, NMR, and mutation studies of two tandem
cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170,
CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both
CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a
more basic groove and directly binds the EExEEY/F motif of the C-terminal
acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that
is furnished with a less positively charged surface only weakly interacts with
the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette
motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle
domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic
tails. These results provide a structural basis for the proposed CLIP-170
copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly
binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating
that EB1 localized at the plus end contributes to CLIP-170 recruitment to the
plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or
nucleation by neutralizing the negative charges of tubulins with the highly
positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds
microtubule, the released zinc knuckle domain may serve to recruit dynein to the
plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide
the structural basis for the specific dynein loading on the microtubule plus end.
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Figure 1.
Fig. 1. Crystal structures of the CAP-Gly-1 and CAP-Gly-2
domains. (a) Domain organization of the CLIP-170 dimer. The
N-terminal CAP-Gly domains directly bind MT/tubulin. This
binding is autoinhibited by the C-terminal zinc knuckle domains,
which also serve to recruit dynein by interacting with
p150^Glued and LIS1. (b) Ribbon drawings of the crystal
structure of the CLIP-170 CAP-Gly-1 domain (Left), electrostatic
potentials on the front molecular surface in the same molecular
orientation (Center), and a 180° rotated image (Right). The
region corresponding to the GKNDG motif is circled with yellow
broken lines. (c) Same figures as in b but for the CLIP-170
CAP-Gly-2 domain. Electrostatic potentials of the front surfaces
of the CAP-Gly domains of p150^Glued (PDB ID, 2HL3) (d), CYLD
(1IXD) (e), and F53.43 (1LPL) (f).
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Figure 3.
Fig. 3. Solution structure of the CAP-Gly-2–peptide
complex. (a) A best-fit superimposition of the final 20
simulated annealing structures of the CLIP-170 CAP-Gly-2 domain
(residues 212–281) bound to the  3-tubulin peptide
(447–451) with the lowest energies is displayed. The CAP-Gly-2
domain backbone (cyan) and selected side chains (blue) are
depicted with the peptide (magenta). (b) Close-up view of the
hydrogen bonds (dotted lines) formed in the complex along the
arrow in a (Right). The side chain of Phe-236 is omitted for
clarity. (c) Pull-down assay of mutated CAP-Gly-2 domains with
the 3-tubulin peptide
suggests a critical role for Asn-253 in peptide binding.
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