Myosin IX: A Single-Headed Processive Motor

The class IX myosin is a member of the myosin superfamily and found in variety of tissues. Myosin IX is quite unique among the myosin superfamily in that the tail region contains a GTPase activating protein (GAP) domain for the small GTP-binding protein, Rho. Recently it was reported that myosin IX shows processive movement that travels on an actin filament for a long distance. This was an intriguing discovery, because myosin IX is a “single-headed†myosin unlike other processive myosins which have “double-headed†structure. It has been thought that “processive†motors walk on their track with their two heads, thus traveling for a long distance. Therefore, it is reasonable to expect that the processive movement of single headed myosin IX is based on the unique feature of myosin IX motor function. In this study, I investigated the mechanism of processive movement of single-headed myosins by analyzing the mechanism of ATPase cycle of myosin IX that is closely correlated with the cross-bridge cycle (the mechanical cycle of actomyosin). In the first part, I performed the transient enzyme kinetic analysis of myosin IX using the motor domain construct to avoid the complexity raised by the presence of the tail domain. It was revealed that the kinetical characteristics of myosin IX ATPase is quite different from other processive myosins. It was particularly notable that the affinity of the weak actin binding state of Myosin IX was extremely high comparing with known myosins. It is thought that the high affinity for actin throughout the ATPase cycle is a major component to explain the processive movement of myosin IX. In the second part of this study, I cloned full length human myosin IX construct to further investigate the regulation of motor activity of myosin IX. It was revealed that the basal ATPase activity but not the actin dependent ATPase activity of myosin IX is inhibited by its tail region. Furthermore full-length myosin IX is regulated by calcium, presumably due to the calcium binding to the CaM light chain. These result suggest that the tail domain serves as a regulatory component of myosin IX

APPROACHING EFFICIENT NANOMOTORS VIA BIOMIMICKING MECHANISMS

Ph.DDOCTOR OF PHILOSOPH

Kinesin-1 mechanical flexibility and motor cooperation

Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation

Simulating the kinesin walk : towards a definitive theory

Dementia is a set of incurable, fatal diseases characterised by irreversible degeneration of the brain. One theory of its cause is the failure of intracellular transport in the axons of the neurons that compose the brain. Kinesin is a key motor transporting vital cargo along the axon. We know that this motor is a bipedal engine stepping forward along a polypeptide track but it is too small and fast for this motion to be observed using current experimental techniques. The stepping detail is therefore open to debate. This study firstly addresses the question of how kinesin steps and secondly pilots a possible method for investigating transport disruption in silico. To investigate the detail of stepping, a program has been designed and built to simulate kinesin traversing its track along a section of axon. The motor is modelled as simple, interacting agents obeying rules abstracted from known chemical and binding properties of its components. The agent-based method has proven useful and efficient on the small scale and has potential for simulating the larger and more complex system of axonal transport. This would enable investigation of transport failure in the context of finding a cure for dementia. A new model of kinesin stepping has been formulated as a consequence of performing virtual experiments using the simulation. Analysis of in vivo and in vitro experimental studies shows that the model accounts for a wide range of published results, explaining many findings. New experiments are suggested to test the model based on its falsifiable predictions. The principal conclusion of this study is that kinesin stepping is rectified Brownian motion.EThOS - Electronic Theses Online ServiceEngineering and Physical Sciences Research Council (EPSRC)GBUnited Kingdo

Molecular basis of the kinematics of the kinesin step

Kinesin is an ATP-dependent cellular transporter that ferries cargos towards the plus-end of a microtubule. Despite significant advances in experiments, which have provided deep insights into the motility of kinesin, the molecular events that occur in a single step have not been fully resolved. In order to provide these details, this thesis develops a structure of the complex between kinesin and microtubule, and devises new simulation methods to probe the stepping kinetics over a wide range of conditions. Hundreds of molecular movies of kinesin walking on the microtubule are generated using coarse-grained simulation methods. Analysis of these movies shows that there are three major stages in the stepping kinetics of kinesin. In addition, an allosteric network within kinesin, responsible for controlling nucleotide release, is identified using microsecond all-atom simulations. These simulations are used to answer two important questions. First, does kinesin move by a "power stroke" or by diffusion? During a single step, the trailing head of the kinesin detaches from the microtubule, passes the microtubule-bound leading head, and attaches to the target binding site 16 nm away. The target binding site, however, is one of eight accessible binding sites on the microtubule. Is it possible that the "power stroke" (a large conformational change) in the leading head, pulls the trailing head into the neighborhood of the target binding site? This remained unclear because the fraction of the 16 nm step associated with the power stroke and diffusion had never been quantified. Second, how does the microtubule accelerate ADP release from kinesin, which is a key step in completing a single step? The ADP binding site of kinesin is more than 1.5 nm away from the microtubule binding surface. Therefore, the microtubule must affect the ADP binding site through an allosteric mechanism. However, the structural basis for transmitting signals through the underlying allosteric network was previously unknown. Analysis of hundreds of kinesin steps generated using coarse-grained simulations showed that the power stroke associated with the docking of the neck linker to the leading head, is responsible for only 4 nm of the 16 nm step, and the remaining 12 nm is covered by diffusion. However, the power stroke in the leading head constrains the diffusion of the trailing head, decreases the probability of side steps, and therefore biases the trailing head, to the target binding site. Additional all-atom simulations of the ADP-kinesin-microtubule complex, revealed a surprisingly simple allosteric network within kinesin that explains the acceleration of ADP release upon microtubule binding. The allosteric network also explains two additional experimental observations on ADP release from kinesin