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Team Publications
Structural Motility
Year of publication 2004
Steven S Rosenfeld, Anne Houdusse, H Lee Sweeney (2004 Dec 8)
Magnesium regulates ADP dissociation from myosin V.
The Journal of biological chemistry : 6072-9
Summary
Processivity in myosin V is mediated through the mechanical strain that results when both
heads bind strongly to an actin filament, and this strain regulates the timing of ADP release.
However, what is not known is which steps that lead to ADP release are affected by this
mechanical strain. Answering this question will require determining which of the several
potential pathways myosin V takes in the process of ADP release and how actin influences
the kinetics of these pathways. We have addressed this issue by examining how magnesium
regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a
model in which actin accelerates the release of ADP from myosin V by reducing the
magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the
structural changes that actin induces in myosin to release phosphate. This effect on
magnesium affinity provides a plausible explanation for how mechanical strain can alter this
actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release
and precedes ADP release. Increasing magnesium concentration to within the physiological
range would thus slow both the ATPase activity and the velocity of movement of this motor.
Pierre-Damien Coureux, H Lee Sweeney, Anne Houdusse (2004 Oct 29)
Three myosin V structures delineate essential features of chemo-mechanical
transduction.
The EMBO journal : 4527-37
Summary
The molecular motor, myosin, undergoes conformational changes in order to convert
chemical energy into force production. Based on kinetic and structural considerations, we
assert that three crystal forms of the myosin V motor delineate the conformational changes
that myosin motors undergo upon detachment from actin. First, a motor domain structure
demonstrates that nucleotide-free myosin V adopts a specific state (rigor-like) that is not
influenced by crystal packing. A second structure reveals an actomyosin state that favors
rapid release of ADP, and differs from the rigor-like state by a P-loop rearrangement.
Comparison of these structures with a third structure, a 2.0 angstroms resolution structure of
the motor bound to an ATP analog, illuminates the structural features that provide
communication between the actin interface and nucleotide-binding site. Paramount among
these is a region we name the transducer, which is composed of the seven-stranded betasheet and associated loops and linkers. Reminiscent of the beta-sheet distortion of the F1ATPase, sequential distortion of this transducer region likely controls sequential release of
products from the nucleotide pocket during force generation.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1
Team Publications
Structural Motility
Carolyn A Moores, Mylène Perderiset, Fiona Francis, Jamel Chelly, Anne Houdusse, Ronald A
Milligan (2004 Jun 18)
Mechanism of microtubule stabilization by doublecortin.
Molecular cell : 833-9
Summary
Neurons undertake an amazing journey from the center of the developing mammalian brain
to the outer layers of the cerebral cortex. Doublecortin, a component of the microtubule
cytoskeleton, is essential in postmitotic neurons and was identified because its mutation
disrupts human brain development. Doublecortin stabilizes microtubules and stimulates their
polymerization but has no homology with other MAPs. We used electron microscopy to
characterize microtubule binding by doublecortin and visualize its binding site. Doublecortin
binds selectively to 13 protofilament microtubules, its in vivo substrate, and also causes
preferential assembly of 13 protofilament microtubules. This specificity was explained when
we found that doublecortin binds between the protofilaments from which microtubules are
built, a previously uncharacterized binding site that is ideal for microtubule stabilization.
These data reveal the structural basis for doublecortin’s binding selectivity and provide
insight into its role in maintaining microtubule architecture in maturing neurons.
Amel Bahloul, Guillaume Chevreux, Amber L Wells, Davy Martin, Jocelyn Nolt, Zhaohui Yang, LiQiong Chen, Noëlle Potier, Alain Van Dorsselaer, Steve Rosenfeld, Anne Houdusse, H Lee
Sweeney (2004 Mar 24)
The unique insert in myosin VI is a structural calcium-calmodulin binding site.
Proceedings of the National Academy of Sciences of the United States of America : 4787-92
Summary
Myosin VI contains an inserted sequence that is unique among myosin superfamily members
and has been suggested to be a determinant of the reverse directionality and unusual
motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds
one calmodulin (CaM) by means of a single “IQ motif”. Using truncations of the myosin VI
protein and electrospray ionization(ESI)-MS, we demonstrate that in fact each myosin VI
head binds two CaMs. One CaM binds to a conventional IQ motif either with or without
calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The
second CaM binds to a unique insertion between the converter region and IQ motif. This
unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the Cterminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide
contribute to the Ca(2+)-CaM binding, as truncations that eliminate elements of the MD alter
CaM binding and allow calcium dissociation. We suggest that the Ca(2+)-CaM bound to the
unique insert represents a structural CaM, and not a calcium sensor or regulatory component
of the motor. This structure is likely an integral part of the myosin VI “converter” region and
repositions the myosin VI “lever arm” to allow reverse direction (minus-end) motility on
actin.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2
Team Publications
Structural Motility
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3