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PDBsum entry 3mjx
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Structural protein
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PDB id
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3mjx
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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 mechanism of pentabromopseudilin inhibition of myosin motor activity.
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Authors
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R.Fedorov,
M.Böhl,
G.Tsiavaliaris,
F.K.Hartmann,
M.H.Taft,
P.Baruch,
B.Brenner,
R.Martin,
H.J.Knölker,
H.O.Gutzeit,
D.J.Manstein.
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Ref.
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Nat Struct Biol, 2009,
16,
80-88.
[DOI no: ]
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PubMed id
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Abstract
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We have identified pentabromopseudilin (PBP) as a potent inhibitor of
myosin-dependent processes such as isometric tension development and unloaded
shortening velocity. PBP-induced reductions in the rate constants for ATP
binding, ATP hydrolysis and ADP dissociation extend the time required per myosin
ATPase cycle in the absence and presence of actin. Additionally, coupling
between the actin and nucleotide binding sites is reduced in the presence of the
inhibitor. The selectivity of PBP differs from that observed with other myosin
inhibitors. To elucidate the binding mode of PBP, we crystallized the
Dictyostelium myosin-2 motor domain in the presence of Mg(2+)-ADP-meta-vanadate
and PBP. The electron density for PBP is unambiguous and shows PBP to bind at a
previously unknown allosteric site near the tip of the 50-kDa domain, at a
distance of 16 A from the nucleotide binding site and 7.5 A away from the
blebbistatin binding pocket.
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Figure 1.
(a) Effect of PBP on the steady-state actin-activated ATPase
activitiy of Dd myosin-5b. Actin-activated ATPase activities in
the absence (  square
) and presence of 25  M
PBP (  down
triangle ) are shown. (b) Concentration-dependence of the
inhibition of the actin-activated ATPase activity of different
myosin isoforms in the presence of 0–150 M
PBP. The semilogarithmic plot shows the concentration dependence
for myosin-1E (o), myosin-2 ( square
), Dd myosin-5b (  )
and chicken myosin-5a ( down
triangle ). The concentrations of PBP required for half-maximal
inhibition (IC[50]) of the different myosin motors were
determined from sigmoidal fits of the data. All parameters are
summarized in Table 1. (c) Fluorescence transients obtained upon
mixing 1 M
Dd myosin-5b with 10 M
mantATP in the absence and presence of 5 M,
25 M,
50 M
and 100 M
PBP. The concentration dependence of the amplitude of the fast
phase is shown in the inset. (d) Rate constants for ATP binding
and hydrolysis were determined by following the time-dependent
changes in the intrinsic protein fluorescence of Dd myosin-5b.
Addition of 25 M
PBP leads to a 14-fold reduction in the apparent second-order
rate constant for ATP binding and a ten-fold reduction of the
rate of ATP hydrolysis (Supplementary Methods online). (e)
PBP-mediated inhibition of chicken myosin-5a in the in vitro
motility assay. The histograms show the average sliding velocity
of rhodamine-phalloidin–labeled actin filaments in the absence
and presence of 10 M
PBP.
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Figure 3.
Myosin residues interacting with PBP are shown as predicted
by molecular modeling for Dd myosin-1E (a), Sc Myo2 (b), Gg
myosin-5a (c) and Dd myosin-5b (d).
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol
(2009,
16,
80-88)
copyright 2009.
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