Inter-head Coordination Research Articles

Computational modeling of dynein motor proteins at work.

Along with various experimental methods, a combination of theoretical and computational methods is essential to explore different length-scale and time-scale processes in the biological system. The functional mechanism of a dynein, an ATP-fueled motor protein, working in a multiprotein complex, involves a wide range of length/time-scale events. It generates mechanical force from chemical energy and moves on microtubules towards the minus end direction while performing a large number of biological processes including ciliary beating, intracellular material transport, and cell division. Like in the cases of other conventional motor proteins, a combination of experimental techniques including X-crystallography, cryo-electron microscopy, and single molecular assay have provided a wealth of information about the mechanochemical cycle of a dynein. Dyneins have a large and complex structural architecture and therefore, computational modeling of different aspects of a dynein is extremely challenging. As the process of dynein movement involves varying length and timescales, it demands, like in experiments, a combination of computational methods covering such a wide range of processes for the comprehensive investigation of the mechanochemical cycle. In this review article, we will summarize how the use of state-of-the-art computational methods can provide a detailed molecular understanding of the mechanochemical cycle of the dynein. We implemented all-atom molecular dynamics simulations and hybrid quantum-mechanics/molecular-mechanics simulations to explore the ATP hydrolysis mechanisms at the primary ATPase site (AAA1) of dynein. To investigate the large-scale conformational changes we employed coarse-grained structure-based molecular dynamics simulations to capture the domain motions. Here we explored the conformational changes upon binding of ATP at AAA1, nucleotide state-dependent regulation of the mechanochemical cycle, and inter-head coordination by inter-head tension. Additionally, implementing a phenomenological theoretical model we explore the force-dependent detachment rate of a motorhead from the microtubule and the principle of multi-dynein cooperation during cargo transport.

Molecular mechanisms of the interhead coordination by interhead tension in cytoplasmic dyneins

Cytoplasmic dyneins play a major role in retrograde cellular transport by moving vesicles and organelles along microtubule filaments. Dyneins are multidomain motor proteins with two heads that coordinate their motion via their interhead tension. Compared with the leading head, the trailing head has a higher detachment rate from microtubules, facilitating the movement. However, the molecular mechanism of such coordination is unknown. To elucidate this mechanism, we performed molecular dynamics simulations on a cytoplasmic dynein with a structure-based coarse-grained model that probes the effect of the interhead tension on the structure. The tension creates a torque that influences the head rotating about its stalk. The conformation of the stalk switches from the α registry to the β registry during the rotation, weakening the binding affinity to microtubules. The directions of the tension and the torque of the leading head are opposite to those of the trailing head, breaking the structural symmetry between the heads. The leading head transitions less often to the β registry than the trailing head. The former thus has a greater binding affinity to the microtubule than the latter. We measured the moment arm of the torque from a dynein structure in the simulations to develop a phenomenological model that captures the influence of the head rotating about its stalk on the differential detachment rates of the two heads. Our study provides a consistent molecular picture for interhead coordination via interhead tension.

A mathematical understanding of how cytoplasmic dynein walks on microtubules.

Cytoplasmic dynein 1 (hereafter referred to simply as dynein) is a dimeric motor protein that walks and transports intracellular cargos towards the minus end of microtubules. In this article, we formulate, based on physical principles, a mechanical model to describe the stepping behaviour of cytoplasmic dynein walking on microtubules from the cell membrane towards the nucleus. Unlike previous studies on physical models of this nature, we base our formulation on the whole structure of dynein to include the temporal dynamics of the individual subunits such as the cargo (for example, an endosome, vesicle or bead), two rings of six ATPase domains associated with diverse cellular activities (AAA+ rings) and the microtubule-binding domains which allow dynein to bind to microtubules. This mathematical framework allows us to examine experimental observations on dynein across a wide range of different species, as well as being able to make predictions on the temporal behaviour of the individual components of dynein not currently experimentally measured. Furthermore, we extend the model framework to include backward stepping, variable step size and dwelling. The power of our model is in its predictive nature; first it reflects recent experimental observations that dynein walks on microtubules using a weakly coordinated stepping pattern with predominantly not passing steps. Second, the model predicts that interhead coordination in the ATP cycle of cytoplasmic dynein is important in order to obtain the alternating stepping patterns and long run lengths seen in experiments.

Examining kinesin processivity within a general gating framework.

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ~1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.

The Winch Model Can Explain both Coordinated and Uncoordinated Stepping of Cytoplasmic Dynein

Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.

The Mechanism of Cytoplasmic Dynein Motility

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. The mechanism of dynein processivity and interhead coordination remain unclear due to its large size (2.6 MDa) and the complexity of its structure. In contrast to kinesins and myosins, we directly observed that dynein heads move independently along the microtubule. Stepping behavior of the heads varies as a function of interhead separation and establishing the basis of high variability in dynein step size. By engineering the mechanical and catalytic properties of the dynein motor domain, we show that a rigid linkage between monomers and dimerization between N-terminal tail domains are not essential for processive movement. Instead, dynein processivity minimally requires the linker domain of one active monomer to be attached to an inert MT tether retaining only the MT-binding domain. The release of a dynein monomer from the MT can be mediated either by nucleotide binding or external load. However, nucleotide dependent release is inhibited when force was applied to the linker domain. Force dependent release is significantly asymmetric, with faster release towards the minus-end. On the basis of these measurements, we developed a model that describes the basis of dynein processivity, directionality and force generation.

The Mechanism of Cytoplasmic Dynein Processivity

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. It is currently unknown how the two catalytic head domains interact and move relative to each other during processive movement. We have tracked the relative positions of both heads with nanometer precision and directly observed that the heads move independently along the microtubule. The heads remain widely separated and the stepping behavior of the heads varies as a function of interhead separation. Consistent with a lack of tight coordination, only one active head is sufficient for processive movement and the active head drags its inactive partner head forward. Only a single active ATPase ring is sufficient for processivity, and the linker swing provides required force to drive minus end directed motion. These results show that dynein is the first dimeric motor that moves processively without interhead coordination, a mechanism fundamentally distinct from hand-over-hand motion of kinesin and myosin.

Cytoplasmic Dynein Moves Through Uncoordinated Stepping of the AAA+ Ring Domains

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos toward the microtubule minus end. How the two catalytic head domains interact and move relative to each other during processive movement is unclear. Here, we tracked the relative positions of both heads with nanometer precision and directly observed the heads moving independently along the microtubule. The heads remained widely separated, and their stepping behavior varied as a function of interhead separation. One active head was sufficient for processive movement, and an active head could drag an inactive partner head forward. Thus, dynein moves processively without interhead coordination, a mechanism fundamentally distinct from the hand-over-hand stepping of kinesin and myosin.

Strain through the neck linker ensures processive runs: a DNA-kinesin hybrid nanomachine study.

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13-amino-acid chain positioned at the C-terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter-head coordination through the neck linker facilitates long-distance walking.

Neck-linker And Neck-coil both contribute to Kinesin Processivity

Kinesins are microtubule-based molecular motors involved in intracellular transport. These motors utilize energy from ATP hydrolysis to ‘walk’ along microtubules and are processive, meaning that they take multiple steps before detaching from the microtubule. The kinesin superfamily consists of 14 families of motors that have diverse structures and diverse cellular functions. A principle question in understanding motor function is: What are the structural determinants of processivity. In contrast to homodimeric motors in the canonical Kinesin-1 family, Kinesin-2 motors have two different head domains and a three amino acid extension in their neck linker. We showed previously that Kinesin-2 motors are slower than Kinesin-1 and are roughly four-fold less processive. These differences could result from biochemical differences in the head domains, reduced inter-head coordination due to differences in the neck-linker domains, or diminished electrostatic interactions between the coiled-coil domain and the microtubule track. To test the influence of the coiled-coil domain, we engineered motors containing the Kinesin-2 head and neck linker domains fused to the Kinesin-1 coiled-coil. Single-molecule fluorescence experiments of GFP-labeled motors showed enhanced processivity compared to Kinesin-2, indicating a role for the coiled-coil in motor processivity. When the Kinesin-1 neck-linker domain was extended by three amino acids, processivity fell by a factor of three, suggesting that the neck-linker domain is an important determinant of processivity. However, shortening the Kinesin-2 neck-linker significantly reduced its processivity, indicating that in a given motor, the head, neck-linker and coiled-coil domains are tuned for optimal motor function.