High resolution structural studies to understand the chemomechanical transduction mechanism of myosin motors and the critical interactions that regulate their cellular location. – How myosin produces force

To understand how motors operate, crystallography allows to visualize at atomic resolution the different states of the motor along its cycle. This information helps design specific functional experiments that will test the new insights provided by structures on the mechanism of the motor.

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 30-36nm, 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 new 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.

We collaborate with Cytokinetics, a pharmaceutical company specialized in the identification of drugs against molecular motors. Myosin VI and myosin X are indeed promising anti-tumoral targets.

We have published the first part of our results that show how different myosin VI walks compared to other motors. We show indeed that it doesn’t use a pure swinging lever arm mechanism but instead its lever arm needs to uncouple from the motor when the lead head binds strongly to actin. It is only after the rear head detaches that recoupling occurs to optimize the propulsion toward the minus end of the actin filament.

Motility is one of the hallmarks of all living organisms. It is an essential function for life that powers cell movement and provides the ability to organize the cellular contents of a cell. Such directed movements are achieved by regulation of cytoskeleton dynamics and by conversion of chemical energy into mechanical energy by specialized molecular motors. Myosins are motor proteins that use ATP to power interactions with actin filaments and create force and directed movements. These molecular machines exert an amazing amount of force considering that they are only a few nanometers across. Using X-ray crystallography, the Structural Motility group at the Curie Institute solve atomic structures to gain understanding on how myosin motors produce force, are regulated and recruited in the cell. Structural information on the multiple states the motor adopt along its motor cycle is essential for understanding how chemical energy is converted into force production. In collaboration with the team of Lee Sweeney Upenn USA, we validate and complete this structural approach with functional studies to test the hypotheses suggested by the visualization of the distinct structural states of the motor. Such an integration of functional and structural studies allows us to be particularly competitive to answer critical questions for myosin function, regulation and recruitment.

Force production by myosins is intimately linked to the sequential conformational changes activated by actin that allow the release of the hydrolysis products, inorganic phosphate (Pi) and then ADP. To understand how myosins produce force, it is thus essential to determine at high resolution the structural state that allows release of Pi from this motor. A new structural state of myosin VI with MgADP bound identified in our laboratory seems to have all the requested properties to correspond to this missing state. For the first time, this state shows how an open “backdoor” could allow release of Pi while the lever arm remains in a primed position. Such a position for the lever arm is indeed critical for this state since it corresponds to a state at the beginning of force production.

Our first aim will focus in structural and functional experiments necessary to understand the nature of the Pi release state, which is at the heart of the mechanism that converts chemical energy into force production. Coupled structural and functional studies will allow to test the hypotheses related to the interpretation of the new myosin VI structure with MgADP bound and will illuminate our current understanding of the rearrangements in the motor essential for the release of Pi from the active site at the beginning of force production. High pressure experiments will also aim at describing how the motor functions under strain.

Our second aim is to describe the structural adaptations and rearrangements in the myosin X motor that are at the basis of its atypical motility. This myosin is indeed able to select bundled actin filaments and thus determine the cellular address towards which it must progress, at the tip of the filopodia. To understand this property, structures of the myosin motor domain in different states and of its atypical lever arm will be obtained. We will also try to determine the structure of the region that allows dimerisation of the two heads of this myosin.
While myosin VI seemed to be very enigmatic a few years ago, our structural studies have allowed to identify the structural adaptations that allows its reverse directionality. We are thus confident that similar structural studies with the myosin X motor will lead to the description of the adaptations necessary for the specific motility function of this myosin. Such variations in the motility mechanism of various members of the myosin superfamily are essential to allow these motors to be perfectly designed for their numerous cellular functions.