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PDBsum entry 2bot
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Contractile protein
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PDB id
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2bot
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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 affinity of the dynein microtubule-Binding domain is modulated by the conformation of its coiled-Coil stalk.
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Authors
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I.R.Gibbons,
J.E.Garbarino,
C.E.Tan,
S.L.Reck-Peterson,
R.D.Vale,
A.P.Carter.
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Ref.
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J Biol Chem, 2005,
280,
23960-23965.
[DOI no: ]
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PubMed id
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Abstract
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The microtubule-binding domain (MTBD) of dynein is separated from the AAA
(ATPase with any other activity) core of the motor by an approximately 15-nm
stalk that is predicted to consist of an antiparallel coiled coil. However, the
structure of this coiled coil and the mechanism it uses to mediate communication
between the MTBD and ATP-binding core are unknown. Here, we sought to identify
the optimal alignment between the hydrophobic heptad repeats in the two strands
of the stalk coiled coil. To do this, we fused the MTBD of mouse cytoplasmic
dynein, together with 12-36 residues of its stalk, onto a stable coiled-coil
base provided by Thermus thermophilus seryl-tRNA synthetase and tested these
chimeric constructs for microtubule binding in vitro. The results identified one
alignment that yielded a protein displaying high affinity for microtubules (2.2
microM). The effects of mutations applied to the MTBD of this construct
paralleled those previously reported (Koonce, M. P., and Tikhonenko, I. (2000)
Mol. Biol. Cell 11, 523-529) for an intact dynein motor unit in the absence of
ATP, suggesting that it resembles the tight binding state of native intact
dynein. All other alignments showed at least 10-fold lower affinity for
microtubules with the exception of one, which had an intermediate affinity.
Based on these results and on amino acid sequence analysis, we hypothesize that
dynein utilizes small amounts of sliding displacement between the two strands of
its coiled-coil stalk as a means of communication between the AAA core of the
motor and the MTBD during the mechanochemical cycle.
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Figure 1.
FIG. 1. Molecular structure of intact cytoplasmic dynein.
The cytoplasmic dynein motor is a dimer containing two identical
heavy chain subunits of molecular mass  520 kDa. The core of the
motor, formed by the C-terminal two-thirds of the heavy chain,
comprises a ring of six AAA ATPase domains, depicted here in
blue and purple. The MTBD (blue) protrudes from the AAA core on
a coiled-coil stalk (gray). The attachment of cargo to the
dynein motor involves light and intermediate chain subunits
(green) that are associated with the N-terminal third of the
heavy chain. This figure was adapted from Ref. 2 with permission.
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Figure 5.
FIG. 5. Structural models of the dynein stalk. A, models of
a section of the dynein stalk in configurations expected to
correspond to those occurring in SRS-22:19 or SRS19:19. CC2 is
shown in surface representation with hydrophobic residues (Val,
Ile, Leu, Ala, Met, Tyr) in the coiled-coil core ("a" and "d"
positions) colored green. CC1 is shown in backbone
representation (gray) with side chains included for Ser-3224
(yellow), Leu-3227 (cyan; with asterisk) and Lys-3230 (magenta).
In passing from the configuration of SRS-22:19 to that of
SRS-19:19, the side chain of Leu-3227 shifts from packing
against one side of CC2 (left) to packing against the other side
(right), potentially by following the hydrophobic-lined groove
formed by Ile-3459, Leu-3463, Val-3466 and Val-3470 in the
surface of CC2 (dashed line). Similar hydrophobic grooves in the
core interface of CC2 occur in most other heptads along the
length of the stalk coiled coil (supplemental material). B,
schematics depicting transverse sections through the coiled-coil
stalk in the region shown in A. Residues in CC1 are colored as
in A to illustrate that Leu-3227 (cyan) shifts from a "d" heptad
position to an "a" position in going between the 22:19 and 19:19
configurations (see also Fig. 2A). Panel A of this figure was
prepared with the program MOLMOL (22). Coordinates of these
models have been deposited in the theoretical models section of
the Protein Data Bank under the identifiers 2BOT (SRS-22:19) and
2BOR (SRS-19:19).
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The above figures are
reprinted
from an Open Access publication published by the ASBMB:
J Biol Chem
(2005,
280,
23960-23965)
copyright 2005.
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