Group leader : Anne Houdusse
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.
The 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.
The 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).
The 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.