9.5 Biomolecular motors

Biomolecular motors are tiny protein machines that convert chemical energy into mechanical work, powering various cellular processes. These nanoscale marvels come in two main types: linear motors that move along tracks, and rotary motors that spin around an axis.

Linear motors like myosin, kinesin, and dynein transport cargo and generate forces by "walking" along filaments. Rotary motors like flagellar motors and ATP synthase produce spinning motions. Both types use ATP hydrolysis to drive conformational changes that enable their mechanical functions.

Types of biomolecular motors

• Biomolecular motors are protein machines that convert chemical energy into mechanical work, enabling various cellular processes such as transport, motility, and force generation
• These motors can be classified based on their structural and functional properties, with the main categories being linear motors and rotary motors

Linear vs rotary motors

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• Linear motors (myosin, kinesin, dynein) move along filamentous tracks (actin filaments or microtubules) in a straight line, typically transporting cargo or generating contractile forces
• Rotary motors (flagellar motors, ATP synthase) generate rotational motion around an axis, often involved in propulsion or energy production
• Linear motors undergo conformational changes that result in a stepping motion, while rotary motors exhibit continuous rotation driven by proton gradients or ATP hydrolysis

Myosin motors

• Myosin motors interact with actin filaments and are best known for their role in muscle contraction (skeletal, cardiac, and smooth muscle)
• Myosin II, the conventional myosin found in muscle cells, forms thick filaments that slide along actin thin filaments, generating contractile forces
• Non-muscle myosins (I, V, VI) are involved in various cellular processes such as cell migration, endocytosis, and organelle transport

Kinesin motors

• Kinesin motors transport cargo (organelles, vesicles, protein complexes) along microtubules, typically in the anterograde direction (from the cell body to the periphery)
• Kinesin-1, the conventional kinesin, has two motor domains that alternate in a hand-over-hand motion, allowing processive movement
• Other kinesin families (kinesin-2, kinesin-13) have specialized functions such as intraflagellar transport or microtubule depolymerization

Dynein motors

• Dynein motors, particularly cytoplasmic dynein, transport cargo along microtubules in the retrograde direction (from the periphery to the cell body)
• Axonemal dyneins are responsible for the beating motion of cilia and flagella, enabling cell motility or fluid flow
• Dynein motors have a complex structure with multiple subunits and accessory proteins that regulate their activity and cargo binding

Flagellar motors

• Bacterial flagellar motors are rotary motors that drive the rotation of flagella, enabling bacterial motility and chemotaxis
• The motor consists of a rotor (MS ring) and a stator (MotA/MotB complexes) that generate torque through proton or sodium ion flux
• Flagellar motors can switch between counterclockwise and clockwise rotation, leading to running or tumbling behavior in bacteria

Structure of biomolecular motors

• Biomolecular motors have a modular structure with distinct domains that enable their function and regulation
• The structural organization of these motors is adapted to their specific roles and the filaments they interact with

Motor domains

• The motor domain is the catalytic core of the motor protein, responsible for ATP hydrolysis and filament binding
• In linear motors (myosin, kinesin, dynein), the motor domain undergoes conformational changes that generate force and movement
• The motor domain of rotary motors (flagellar motors) is part of the rotor and interacts with the stator to generate torque

Neck regions

• The neck region connects the motor domain to the tail domain and plays a crucial role in the mechanical properties of the motor
• In myosin motors, the neck region consists of a long α-helical lever arm that amplifies the small conformational changes in the motor domain
• Kinesin and dynein motors have shorter neck linkers that transmit the conformational changes to the tail domain

Tail domains

• The tail domain is responsible for cargo binding, dimerization, and regulation of motor activity
• Myosin tails form coiled-coil dimers and can self-assemble into thick filaments (myosin II) or bind to cargo adaptors (non-muscle myosins)
• Kinesin tails often contain light chain binding sites and can interact with various cargo adaptors or regulatory proteins
• Dynein tails are associated with multiple subunits and accessory proteins that modulate their activity and cargo specificity

Structural adaptations for function

• The structural features of biomolecular motors are tailored to their specific functions and the filaments they interact with
• Myosin motors have a prominent lever arm that enables large step sizes (5-10 nm) and force generation, suitable for muscle contraction and cargo transport
• Kinesin motors have a compact structure with a short neck linker, allowing efficient hand-over-hand motion and long-distance transport along microtubules
• Dynein motors have a unique AAA+ ring structure in their motor domain, which enables a large power stroke and adaptability to various cellular functions
• Flagellar motors have a complex ring structure (rotor) and multiple stator units that generate high torque and enable rapid rotation for bacterial swimming

Mechanisms of motion

• Biomolecular motors convert the chemical energy of ATP hydrolysis into mechanical work through a series of conformational changes and filament interactions
• The specific mechanisms of motion vary between different types of motors but share some common principles

ATP hydrolysis

• ATP hydrolysis is the primary energy source for most biomolecular motors, providing the necessary free energy for conformational changes and motion
• The motor domain contains a nucleotide-binding pocket that catalyzes the hydrolysis of ATP to ADP and inorganic phosphate (Pi)
• The release of the hydrolysis products (ADP and Pi) is often coupled to the force-generating conformational changes in the motor

Conformational changes

• ATP hydrolysis and product release induce conformational changes in the motor domain, which are amplified and transmitted to the neck and tail regions
• In myosin motors, ATP binding causes the dissociation of the motor domain from actin, followed by a lever arm swing upon ATP hydrolysis and Pi release
• Kinesin motors undergo a neck linker docking and undocking cycle, which is coupled to ATP hydrolysis and results in a hand-over-hand stepping motion
• Dynein motors exhibit a power stroke mechanism, where ATP hydrolysis in the AAA+ ring leads to a large-scale conformational change and displacement of the microtubule-binding domain

Binding and release of filaments

• The cyclic binding and release of the motor domains to their respective filaments (actin or microtubules) is essential for the stepping motion and force generation
• Myosin motors have a high affinity for actin in the ADP-bound state and a low affinity in the ATP-bound state, allowing for the attachment-detachment cycle
• Kinesin and dynein motors have a similar nucleotide-dependent affinity for microtubules, with strong binding in the ATP-bound state and weak binding in the ADP-bound state
• The coordination of filament binding and release between the two motor domains enables processive movement and cargo transport

Stepping patterns

• Linear motors exhibit distinct stepping patterns that reflect their mechanochemical cycles and the coordination between the motor domains
• Myosin V and VI motors have a hand-over-hand stepping pattern, where the two motor domains alternate in leading and trailing positions, resulting in a large step size (36 nm for myosin V)
• Kinesin-1 also follows a hand-over-hand mechanism, with a step size of 8 nm that matches the distance between adjacent tubulin dimers on the microtubule
• Cytoplasmic dynein has a more complex stepping pattern, with variable step sizes and occasional backward steps, reflecting its unique AAA+ ring structure and flexibility

Processivity of motors

• Processivity refers to the ability of a motor to take multiple steps along its filament track before dissociating
• Highly processive motors, such as myosin V and kinesin-1, can take hundreds of steps before detaching, enabling efficient long-distance transport of cargo
• Less processive motors, like muscle myosin II, take only a few steps before dissociating, which is suitable for their role in generating contractile forces
• Processivity is determined by the coordination between the motor domains, the duty ratio (fraction of time spent in the strongly bound state), and the affinity for the filament track

Cellular functions

• Biomolecular motors play crucial roles in various cellular processes, enabling the spatial organization and dynamics of the cell
• The specific functions of motors are determined by their structural properties, regulation, and the cellular context in which they operate

Intracellular transport

• Kinesin and dynein motors are the primary drivers of intracellular transport along microtubules, which serve as highways for long-distance cargo movement
• Kinesins generally transport cargo (organelles, vesicles, protein complexes) in the anterograde direction (from the cell body to the periphery), while dyneins mediate retrograde transport (from the periphery to the cell body)
• This bidirectional transport is essential for the proper distribution of cellular components, such as the delivery of synaptic vesicles in neurons or the trafficking of endosomes and lysosomes

Muscle contraction

• Myosin II motors are the key force generators in muscle contraction, working in concert with actin filaments to produce mechanical work
• In skeletal muscle, myosin thick filaments and actin thin filaments form the sarcomere, the basic contractile unit of the muscle fiber
• The cyclic interaction between myosin heads and actin, driven by ATP hydrolysis, results in the sliding of the filaments and the shortening of the sarcomere, leading to muscle contraction

Cell division

• Kinesin and dynein motors play essential roles in the formation and function of the mitotic spindle during cell division
• Kinesin-5 motors (e.g., Eg5) generate outward forces that push the spindle poles apart, while kinesin-14 motors (e.g., HSET) provide inward forces that counterbalance the spindle
• Dynein motors, in association with the dynactin complex, focus the microtubule minus ends at the spindle poles and help position the spindle through cortical anchoring

Ciliary and flagellar movement

• Axonemal dyneins are responsible for the beating motion of cilia and flagella, which are essential for cell motility and fluid flow
• The coordinated activity of dynein motors, attached to the microtubule doublets of the axoneme, generates the bending and oscillatory motion of these organelles
• Kinesin-2 motors, such as the heterotrimeric KIF3 complex, are involved in intraflagellar transport (IFT), which is necessary for the assembly and maintenance of cilia and flagella

Bacterial motility

• Bacterial flagellar motors drive the rotation of flagella, enabling bacterial swimming and chemotaxis
• The motor, embedded in the cell membrane, consists of a rotor (MS ring) and a stator (MotA/MotB complexes) that generate torque through proton or sodium ion flux
• The switching between counterclockwise and clockwise rotation of the motor, regulated by the chemotaxis signaling pathway, results in the alternation between running and tumbling behavior, allowing bacteria to navigate their environment

Regulation of motor activity

• The activity and function of biomolecular motors are tightly regulated to ensure their proper spatial and temporal control within the cell
• Various regulatory mechanisms, involving signaling pathways, post-translational modifications, and motor-associated proteins, modulate the activity, localization, and cargo binding of motors

Calcium signaling

• Calcium ions (Ca2+) are universal second messengers that regulate the activity of many biomolecular motors, particularly myosin motors
• In muscle cells, the binding of Ca2+ to the troponin complex triggers a conformational change that exposes the myosin-binding sites on actin, allowing for muscle contraction
• Some non-muscle myosins, such as myosin V, have calmodulin light chains that bind Ca2+ and modulate the motor's activity and cargo interaction

Phosphorylation

• Phosphorylation is a common post-translational modification that regulates the activity and function of biomolecular motors
• Kinases and phosphatases add or remove phosphate groups from specific residues on the motor or its associated proteins, altering their conformation, interaction, or localization
• For example, the phosphorylation of the myosin regulatory light chain by myosin light chain kinase (MLCK) enhances the motor activity and force production in smooth muscle and non-muscle cells

Cargo binding and release

• The binding and release of cargo are regulated by various mechanisms to ensure the specificity and timing of transport
• Cargo adaptor proteins, such as the dynactin complex for dynein or the kinesin light chains for kinesin-1, mediate the interaction between the motor and its cargo
• Rab GTPases, which are molecular switches that cycle between GTP-bound (active) and GDP-bound (inactive) states, play a key role in the recruitment and activation of motor-cargo complexes

Motor-associated proteins

• A wide range of motor-associated proteins, including scaffolding proteins, regulatory subunits, and cofactors, modulate the activity and function of biomolecular motors
• Dynein activity is regulated by a complex network of associated proteins, such as dynactin, LIS1, and NudE/NudEL, which affect its processivity, force generation, and cargo interaction
• Kinesin-binding protein (TRAK/Milton) and mitochondrial Rho GTPase (Miro) form a complex that links kinesin motors to mitochondria and regulates their transport in response to calcium signals

Biophysical properties

• Biomolecular motors exhibit remarkable biophysical properties that enable them to perform their cellular functions efficiently
• These properties, such as force generation, velocity, and efficiency, are determined by the structural and mechanochemical characteristics of the motors

Force generation

• Biomolecular motors generate force through the conformational changes induced by ATP hydrolysis and filament interaction
• The force generated by a single motor is typically in the piconewton (pN) range, with myosin II motors producing forces of 3-4 pN and kinesin-1 motors generating forces of 5-7 pN
• The collective action of multiple motors can generate much higher forces, as observed in muscle contraction or the movement of large organelles

Velocity and speed

• The velocity of biomolecular motors refers to the speed at which they move along their filament track
• Different motors exhibit varying velocities, depending on their mechanochemical cycle and the cellular context
• Kinesin-1 motors have a velocity of about 800 nm/s, while myosin V motors move at a slower pace of 200-400 nm/s, reflecting their different stepping patterns and processivity

Stall force

• The stall force is the maximum force that a motor can generate before it stops moving, representing the balance between the motor's force output and the opposing load
• The stall force provides insight into the force-generating capacity and the mechanochemical coupling of the motor
• Kinesin-1 has a stall force of about 7 pN, while muscle myosin II can generate a stall force of 3-4 pN per motor head

Duty ratio

• The duty ratio is the fraction of time that a motor spends in the strongly bound state (attached to its filament) during its mechanochemical cycle
• Motors with a high duty ratio (>0.5), such as myosin V and kinesin-1, are highly processive and can take multiple steps before dissociating from the filament
• Low duty ratio motors, like muscle myosin II, spend more time in the weakly bound state and are less processive, suitable for their role in generating contractile forces

Thermodynamic efficiency

• Biomolecular motors convert the chemical energy of ATP hydrolysis into mechanical work with remarkable efficiency
• The thermodynamic efficiency of a motor is the ratio of the work performed to the free energy input from ATP hydrolysis
• Kinesin-1 motors have a thermodynamic efficiency of about 50%, while muscle myosin II motors have an efficiency of 30-40%, indicating their optimized energy utilization for their respective functions

Experimental techniques

• The study of biomolecular motors relies on a range of experimental techniques that enable the measurement of their biophysical properties and the observation of their behavior at the single-molecule level
• These techniques have greatly advanced our understanding of motor function and regulation, providing insights into their mechanochemical mechanisms and cellular roles

Optical tweezers

• Optical tweezers use focused laser beams to trap and manipulate small dielectric particles, such as polystyrene beads, which can be attached to biomolecular motors or their filament tracks
• By measuring the displacement of the trapped particle from the center of the laser focus, the force generated by the motor can be determined with high precision (in the piconewton range)
• Optical tweezers have been used to study the stepping behavior, force-velocity relationships, and load-dependent properties of various motors, such as kinesin-1 and myosin V

Single-molecule fluorescence

• Single-molecule fluorescence techniques, such as total internal reflection fluorescence (TIRF) microscopy, allow the visualization of individual motor proteins labeled with fluorescent dyes or genetically encoded fluorescent proteins
• By tracking the movement of fluorescently labeled motors along their filament tracks, the stepping dynamics, processivity, and velocity of the motors can be measured with nanometer precision
• Single-molecule fluorescence has also been used to study the coordination between motor domains, the binding and release of