Motilité structurale

Publications de l’équipe

Année de publication : 2012

Julie Ménétrey, Tatiana Isabet, Virginie Ropars, Monalisa Mukherjea, Olena Pylypenko, Xiaoyan Liu, Javier Perez, Patrice Vachette, H Lee Sweeney, Anne M Houdusse (2012 Mar 19)

Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI.

Molecular cell : 75-86 : DOI : 10.1016/j.molcel.2012.07.034 En savoir plus
Résumé

Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

Replier

Année de publication : 2011

Paola Llinas, Olena Pylypenko, Tatiana Isabet, Monalisa Mukherjea, H Lee Sweeney, Anne M Houdusse (2011 Dec 17)

How myosin motors power cellular functions: an exciting journey from structure to function: based on a lecture delivered at the 34th FEBS Congress in Prague, Czech Republic, July 2009.

The FEBS journal : 551-62 : DOI : 10.1111/j.1742-4658.2011.08449.x En savoir plus
Résumé

Molecular motors such as myosins are allosteric enzymes that power essential motility functions in the cell. Structural biology is an important tool for deciphering how these motors work. Myosins produce force upon the actin-driven conformational changes controlling the sequential release of the hydrolysis products of ATP (Pi followed by ADP). These conformational changes are amplified by a ‘lever arm’, which includes the region of the motor known as the converter and the adjacent elongated light chain binding region. Analysis of four structural states of the motor provides a detailed understanding of the rearrangements and pathways of communication in the motor that are necessary for detachment from the actin track and repriming of the motor. However, the important part of the cycle in which force is produced remains enigmatic and awaits new high-resolution structures. The value of a structural approach is particularly evident from clues provided by the structural states of the reverse myosin VI motor. Crystallographic structures have revealed that rearrangements within the converter subdomain occur, which explains why this myosin can produce a large stroke in the opposite direction to all other myosins, despite a very short lever arm. By providing a detailed understanding of the motor rearrangements, structural biology will continue to reveal essential information and help solve current enigma, such as how actin promotes force production, how motors are tuned for specific cellular roles or how motor/cargo interactions regulate the function of myosin in the cell.

Replier
Stephanie Guzik-Lendrum, Attila Nagy, Yasuharu Takagi, Anne Houdusse, James R Sellers (2011 Apr 19)

Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties.

The Journal of biological chemistry : 21755-66 : DOI : 10.1074/jbc.M111.218669 En savoir plus
Résumé

The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-ΔPDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-ΔPDZ proteins bound to actin with K(d) values of 2.61 and 1.04 μM, respectively, but only about 50-75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein.

Replier
Olena Pylypenko, Lin Song, Gaelle Squires, Xiaoyan Liu, Alan B Zong, Anne Houdusse, H Lee Sweeney (2011 Feb 1)

Role of insert-1 of myosin VI in modulating nucleotide affinity.

The Journal of biological chemistry : 11716-23 : DOI : 10.1074/jbc.M110.200626 En savoir plus
Résumé

Myosin VI is unique in its directionality among myosin superfamily members and also displays a slow and strain-dependent rate of ATP binding that allows for gating between its heads. In this study we demonstrate that leucine 310 is positioned by a class VI-specific insert, insert-1, so as to account for the selective hindrance of ATP versus ADP binding. Mutation of leucine 310 to glycine removes all influence of insert-1 on ATP binding. Furthermore, by analyzing myosin VI structures with either leucine 310 substituted to a glycine or complete removal of insert-1, we conclude that nucleotides may initially bind to myosin by their purine rings before docking their phosphate moieties. Otherwise, insert-1 could not exert a differential influence on ATP versus ADP binding.

Replier

Année de publication : 2010

Olena Pylypenko, Anne M Houdusse (2010 Dec 24)

Essential « ankle » in the myosin lever arm.

Proceedings of the National Academy of Sciences of the United States of America : 5-6 : DOI : 10.1073/pnas.1017676108 En savoir plus
Résumé

Replier
Franck J Fourniol, Charles V Sindelar, Béatrice Amigues, Daniel K Clare, Geraint Thomas, Mylène Perderiset, Fiona Francis, Anne Houdusse, Carolyn A Moores (2010 Oct 27)

Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution.

The Journal of cell biology : 463-70 : DOI : 10.1083/jcb.201007081 En savoir plus
Résumé

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

Replier
Amel Bahloul, Vincent Michel, Jean-Pierre Hardelin, Sylvie Nouaille, Sylviane Hoos, Anne Houdusse, Patrick England, Christine Petit (2010 Jul 20)

Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids.

Human molecular genetics : 3557-65 : DOI : 10.1093/hmg/ddq271 En savoir plus
Résumé

Cadherin-23 is a component of early transient lateral links of the auditory sensory cells’ hair bundle, the mechanoreceptive structure to sound. This protein also makes up the upper part of the tip links that control gating of the mechanoelectrical transduction channels. We addressed the issue of the molecular complex that anchors these links to the hair bundle F-actin core. By using surface plasmon resonance assays, we show that the cytoplasmic regions of the two cadherin-23 isoforms that do or do not contain the exon68-encoded peptide directly interact with harmonin, a submembrane PDZ (post-synaptic density, disc large, zonula occludens) domain-containing protein, with unusually high affinity. This interaction involves the harmonin Nter-PDZ1 supramodule, but not the C-terminal PDZ-binding motif of cadherin-23. We establish that cadherin-23 directly binds to the tail of myosin VIIa. Moreover, cadherin-23, harmonin and myosin VIIa can form a ternary complex, which suggests that myosin VIIa applies tension forces on hair bundle links. We also show that the cadherin-23 cytoplasmic region, harmonin and myosin VIIa interact with phospholipids on synthetic liposomes. Harmonin and the cytoplasmic region of cadherin-23, both independently and as a binary complex, can bind specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), which may account for the role of this phospholipid in the adaptation of mechanoelectrical transduction in the hair bundle. The distributions of cadherin-23, harmonin, myosin VIIa and PI(4,5)P(2) in the growing and mature auditory hair bundles as well as the abnormal locations of harmonin and myosin VIIa in cadherin-23 null mutant mice strongly support the functional relevance of these interactions.

Replier
H Lee Sweeney, Anne Houdusse (2010 May 19)

Myosin VI rewrites the rules for myosin motors.

Cell : 573-82 : DOI : 10.1016/j.cell.2010.04.028 En savoir plus
Résumé

Myosin VI is the only type of myosin motor known to move toward the minus ends of actin filaments. This reversal in the direction of its movement is in part a consequence of the repositioning of its lever arm. In addition, myosin VI has a number of other specialized structural and functional adaptations that optimize performance of its unique cellular roles. Given that other classes of myosins may share some of these features, understanding the design principles of myosin VI will help guide the study of the functions of myosins that adopt similar strategies.

Replier
H Lee Sweeney, Anne Houdusse (2010 Mar 3)

Structural and functional insights into the Myosin motor mechanism.

Annual review of biophysics : 539-57 : DOI : 10.1146/annurev.biophys.050708.133751 En savoir plus
Résumé

The general structural features of the motor region of myosin superfamily members are now well established, as is a subset of the structural and kinetic transitions of the actin-myosin catalytic cycle. Not yet visualized are the structural rearrangements triggered by actin binding that are coupled to force generation and product release. In this review we describe the recent progress in understanding these missing components of the mechanism of chemomechanical transduction by myosin motors. These insights come from a combination of kinetic and single-molecule studies on multiple classes of myosins, with additional insights from contracting muscle fibers. These recent studies have explored the effects of intermediate and high loads on the kinetics of the actin-bound myosin state transitions. We also describe studies that delineate how some classes of myosin motors are adapted for processive movement on actin.

Replier

Année de publication : 2009

Humberto Fernandes, Edward Franklin, Rosario Recacha, Anne Houdusse, Bruno Goud, Amir R Khan (2009 Sep 17)

Structural aspects of Rab6-effector complexes.

Biochemical Society transactions : 1037-41 : DOI : 10.1042/BST0371037 En savoir plus
Résumé

The small GTPase Rab6 regulates vesicle trafficking at the level of Golgi. Recently, the crystal structures of Rab6 in complexes with two unrelated effectors have been determined. The structure of Rab6a-GTP in complex with a 378-residue internal fragment of the effector Rab6IP1 (Rab6-interacting protein 1) has been solved. In addition, the structure of Rab6 with the golgin, GCC185, has also been determined. In both complexes, two alpha-helices from the effector mediate binding to switch I, switch II and the interswitch region of Rab6. Comparisons of the complexes reveal significant conformational changes in the conserved hydrophobic triad of Rab6. Thus conformational flexibility in the triad mediates recognition of compositionally distinct alpha-helical coiled coils, providing a rationale for the promiscuity of Rab6 in effector recruitment.

Replier
Monalisa Mukherjea, Paola Llinas, HyeongJun Kim, Mirko Travaglia, Daniel Safer, Julie Ménétrey, Clara Franzini-Armstrong, Paul R Selvin, Anne Houdusse, H Lee Sweeney (2009 Aug 12)

Myosin VI dimerization triggers an unfolding of a three-helix bundle in order to extend its reach.

Molecular cell : 305-15 : DOI : 10.1016/j.molcel.2009.07.010 En savoir plus
Résumé

Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.

Replier
Tatiana Isabet, Guillaume Montagnac, Karine Regazzoni, Bertrand Raynal, Fatima El Khadali, Patrick England, Michel Franco, Philippe Chavrier, Anne Houdusse, Julie Ménétrey (2009 Aug 1)

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

The EMBO journal : 2835-45 : DOI : 10.1038/emboj.2009.209 En savoir plus
Résumé

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4’s specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

Replier
Anne Houdusse, Andrew P Carter (2009 Feb 11)

Dynein swings into action.

Cell : 395-6 : DOI : 10.1016/j.cell.2009.01.026 En savoir plus
Résumé

Motor proteins, such as dynein, use chemical energy from ATP hydrolysis to move along the cytoskeleton. Roberts et al. (2009) now describe the arrangement of subdomains in the motor domain of dynein and propose a model for how these regions function together in force generation.

Replier
Rosario Recacha, Annick Boulet, Florence Jollivet, Solange Monier, Anne Houdusse, Bruno Goud, Amir R Khan (2009 Jan 15)

Structural basis for recruitment of Rab6-interacting protein 1 to Golgi via a RUN domain.

Structure (London, England : 1993) : 21-30 : DOI : 10.1016/j.str.2008.10.014 En savoir plus
Résumé

Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 angstroms resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment.

Replier

Année de publication : 2008

Emmanuel Derivery, Jenny Fink, Davy Martin, Anne Houdusse, Matthieu Piel, Theresia E Stradal, Daniel Louvard, Alexis Gautreau (2008 Jun 19)

Free Brick1 is a trimeric precursor in the assembly of a functional wave complex.

PloS one : e2462 : DOI : 10.1371/journal.pone.0002462 En savoir plus
Résumé

The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form.

Replier