imprimer la page

-A +A

Structural motility

Keywords : intracellular transport, crystallography, structure/function, motility, molecular motors, motor mechanism, structural biology, allostery

Group leader : Anne Houdusse

Structural motility

Read the scientific activity report.

Intracellular motility is one of the hallmarks of all living organisms. Cellular components such as membrane transport vesicles and organelles move in specific directions within the cell either due to the dynamic assembly and disassembly of the cytoskeleton or by the conversion of chemical energy into mechanical energy by specialised molecular motor proteins. Myosins are one family of these motor proteins; they use cellular ATP to power interactions with actin filaments (F-actin), so generating force and directed movement.

The Structural Motility group at the Institut Curie uses X-ray crystallography to solve atomic structures that help us understand how myosin motors produce force, how their activity is regulated and how they are recruited to specific vesicles or organelles. Structural information about the various states the motor protein adopts during a cycle of ATP binding, hydrolysis and release is essential to understand how chemical energy is converted into force production. In collaboration with a large team of myosin experts co-ordinated by Dr Lee Sweeney (University of Pennsylvania, USA), we also validate and complement this structural approach with functional studies to test hypotheses suggested by visualising the distinct structural states of the motor. This integration of functional and structural studies allows us to answer crucial questions about myosin's function, regulation and recruitment.

Fig. 1Fig. 1The myosin power stroke
The catalytic domain of myosin (purple) binds to F-actin (yellow) in the pre-powerstroke conformation, which places the lever arm in the primed position (red). After force generation, the motor adopts a rigor conformation with the lever arm down (blue).

Actin-driven conformational changes in the myosin motor lead to the sequential release of the hydrolysis products of ATP - inorganic phosphate (Pi) followed by ADP - to produce a force that moves the myosin relative to the F-actin. These conformational changes in the motor are amplified by a ‘lever arm' - an extended α-helix composed of IQ motifs, which act as binding sites for the calmodulin-like regulatory myosin light chains. The lever arm is attached to a region of the motor known as the converter that is believed to amplify and convert the movements resulting from actin-coupled changes in motor conformation. This results in a swing of the lever arm roughly parallel to the actin filament, known as the myosin powerstroke (Fig. 1). The powerstroke begins when myosin, with Pi and ADP bound at its active site, binds to F-actin (the pre-powerstroke state with the converter in the ‘primed' position) and ends when the hydrolysis products are released (the rigor state with the converter in the ‘down' position). This ‘swinging lever arm' mechanism predicts that the powerstroke is proportional to the length of the lever arm; this has been shown to be the case for myosin II and myosin V, but it is not yet clear whether it applies to myosin VI.

Fig. 2Fig. 2The structure of nucleotide-free myosin V reveals the rigor conformation
The structure of the motor domain of myosin V in the rigor-like state (left) shows that the nucleotide-binding elements adopt new conformations that reduce the affinity for the nucleotide compared to the conformation seen in the pre-powerstroke state. The new conformation of the central β-sheet (blue and grey) is linked to the closure of the 50 κDa cleft that separates the U50 and L50 subdomains. On the right, the interface of the myosin molecule with actin (black arrow) is shown. Both the U50 and L50 subdomains contribute to the surface of interaction. The myosin V interface (blue) is compared to that found for weak actin-binding states of myosin (red), after superimposition of their L50 subdomains. Note that the U50 subdomain cannot reach F-actin when the cleft is open (red) but rotates to come closer to F-actin in rigor (blue).

Our crystal structure of myosin V without nucleotide, published in 2003, described for the first time the high-resolution structure of the rigor state of myosin. By crystallising the same motor with a Mg2+-ATP analogue, we demonstrated at atomic resolution the rearrangements in the motor domain that correspond to its detachment from F-actin (Fig. 2).

Fig. 3Fig. 3The structure of nucleotide-free myosin VI compared
Note the difference in the position of the lever arm of myosin VI (right; IQ motif, pale blue) compared to that of myosin V (left; IQ motif, pale blue) due to the myosin VI insert (purple) and its bound molecule of Ca2+-calmodulin (4Ca2+CaM, pink). The lever arm of myosin V is directed towards the plus-end of the actin filament at the end of the powerstroke whereas the lever arm of myosin VI is directed towards the minus-end of the actin filament.

More recently, we also solved the structure of myosin VI in the rigor state (Fig. 3). Myosin VI is a minus-end directed motor: it works in the opposite direction to other myosins, moving towards the so-called ‘minus-end' of F-actin. Among the myosins, it has a unique mechanism, only poorly understood, that allows it to take several steps on an actin filament without detaching from it. Surprisingly, these steps are similar in size to those of myosin V, even though the lever arm of myosin VI contains only one IQ motif, whereas that of myosin V contains six. Our myosin VI rigor state structure reveals that a stretch of 39 amino acid residues (K771-K809), not present in other myosin types, both bends around the converter and binds a calmodulin that interacts with the converter (Fig. 3). The result is a ~120º repositioning of the lever arm relative to other myosins, which explains its reverse directionality. To account for the large powerstroke of myosin VI, however, the pre-powerstroke state of myosin VI must differ from that of plus-end directed myosins.

Our current research focuses on the structures of other biochemical states of myosin VI, the most enigmatic of all myosins. We need to describe other states of the ATPase cycle to understand fully how this ‘reverse' motor moves and produces force. Preliminary data on the pre-powerstroke state reveal, unexpectedly, that a conformational change within the converter takes place; we must now confirm that this novel conformation of the converter exists in other structures and/or find evidence for it from functional studies. In addition, we are using several innovative approaches to gain insights into the state that allows Pi release, which is at the heart of force production by myosin. Finally, because no structural information is yet available about how myosin recognises its cargo, we aim to obtain atomic structures of the full lever arm of myosin VI and of the cargo domain of various myosins alone or bound to cellular partners.

Key publications

  • Year of publication : 2012

  • 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.

  • 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.

  • Year of publication : 2010

  • 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.

  • 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.

  • Year of publication : 2009

  • 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.

  • 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.

  • Year of publication : 2008

  • Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP.

  • Year of publication : 2007

  • Due to a unique addition to the lever arm-positioning region (converter), class VI myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized myosin classes. However, the large size of the myosin VI lever arm swing (powerstroke) cannot be explained by our current view of the structural transitions that occur within the myosin motor. We have solved the crystal structure of a fragment of the myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it provides a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed myosin classes.

  • Sweeney LH, Houdusse A (2007 Feb) What can myosin VI do in cells?

    Current opinion in cell biology, 19(1): 57-66

    The recently solved structure of the myosin VI motor demonstrates that the unique insert at the end of the motor is responsible for the reversal of the normal myosin directionality. A second class-specific insert near the nucleotide-binding pocket contributes to myosin VI's unique kinetic tuning, allowing it to function either as an actin-based transporter or as an anchoring protein. Recent biochemical and biophysical studies have shown that the native molecule can form dimers upon clustering, and cell biological studies have demonstrated that it clearly does play both transport and anchoring roles in cells. These mechanistic insights allow us to speculate on how unusual aspects of myosin VI structure and function allow it to fill unique niches in cells.

  • Year of publication : 2006

  • A 2.5-A resolution structure of calcium-free calmodulin (CaM) bound to the first two IQ motifs of the murine myosin V heavy chain reveals an unusual CaM conformation. The C-terminal lobe of each CaM adopts a semi-open conformation that grips the first part of the IQ motif (IQxxxR), whereas the N-terminal lobe adopts a closed conformation that interacts more weakly with the second part of the motif (GxxxR). Variable residues in the IQ motif play a critical role in determining the precise structure of the bound CaM, such that even the consensus residues of different motifs show unique interactions with CaM. This complex serves as a model for the lever arm region of many classes of unconventional myosins, as well as other IQ motif-containing proteins such as neuromodulin and IQGAPs.

  • Year of publication : 2005

  • Here we solve a 2.4-A structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective 'lever arm' of myosin VI, which includes a second calmodulin bound to an 'IQ motif', towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.

  • Year of publication : 2004

  • 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 beta-sheet and associated loops and linkers. Reminiscent of the beta-sheet distortion of the F1-ATPase, sequential distortion of this transducer region likely controls sequential release of products from the nucleotide pocket during force generation.

  • 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.

  • Sweeney LH, Houdusse A (2004 Dec 29) The motor mechanism of myosin V: insights for muscle contraction.

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 359(1452): 1829-41

    It is 50 years since the sliding of actin and myosin filaments was proposed as the basis of force generation and shortening in striated muscle. Although this is now generally accepted, the detailed molecular mechanism of how myosin uses adenosine triphosphate to generate force during its cyclic interaction with actin is only now being unravelled. New insights have come from the unconventional myosins, especially myosin V. Myosin V is kinetically tuned to allow movement on actin filaments as a single molecule, which has led to new kinetic, mechanical and structural data that have filled in missing pieces of the actomyosin-chemo-mechanical transduction puzzle.

  • Holmes K C, Schröder R R, Sweeney H L, Houdusse A (2004 Dec 29) The structure of the rigor complex and its implications for the power stroke.

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 359(1452): 1819-28

    Decorated actin provides a model system for studying the strong interaction between actin and myosin. Cryo-energy-filter electron microscopy has recently yielded a 14 A resolution map of rabbit skeletal actin decorated with chicken skeletal S1. The crystal structure of the cross-bridge from skeletal chicken myosin could not be fitted into the three-dimensional electron microscope map without some deformation. However, a newly published structure of the nucleotide-free myosin V cross-bridge, which is apparently already in the strong binding form, can be fitted into the three-dimensional reconstruction without distortion. This supports the notion that nucleotide-free myosin V is an excellent model for strongly bound myosin and allows us to describe the actin-myosin interface. In myosin V the switch 2 element is closed although the lever arm is down (post-power stroke). Therefore, it appears likely that switch 2 does not open very much during the power stroke. The myosin V structure also differs from the chicken skeletal myosin structure in the nucleotide-binding site and the degree of bending of the backbone beta-sheet. These suggest a mechanism for the control of the power stroke by strong actin binding.

  • 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 C-terminal 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.

  • Year of publication : 2003

  • The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force. Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core, the structural details of the rearrangement in myosin that drive the lever arm motion on actin attachment are unknown. Motivated by kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we obtained a 2.0 A structure of a myosin V fragment. Here we reveal a conformation of myosin without bound nucleotide. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule, and reveal elements of the structural communication between the actin-binding interface and nucleotide-binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

  • Year of publication : 2001

  • High-resolution structures of the motor domain of myosin II and lower resolution actin-myosin structures have led to the "swinging lever arm" model for myosin force generation. The available kinetic data are not all easily reconciled with this model and understanding the final details of the myosin motor mechanism must await actin-myosin co-crystals. The observation that myosin can populate multiple states in the absence of actin has nonetheless led to significant insights. The currently known myosin structures correspond to defined kinetic states that bind weakly (K(d)>microM) to actin. It is possible that the myosin lever arm could complete its swing before strong binding to actin and force generation--a process that would correspond, in the absence of load, to a Brownian ratchet. We further suggest that, under load, internal springs within the myosin head could decouple force generation and lever arm movement.