Fundamentals of Molecular Biology. Part 1: Molecular Biology of the Cell - A. N. Ogurtsov 2011

Molecular Basis of Protein Function
Molecular Motors

The mechanical translocation of Cells is driven by specialized Enzymes known as molecular motors.

Molecular motors generate either linear or rotational movement (Tables 6 and 7). These mechanochemical enzymes convert the energy of ATP Hydrolysis or an ion gradient into mechanical force.

Some "motor" Proteins are components of macromolecular assemblies, such as the ribosome, whereas others, which move along cytoskeletal filaments, are not. The latter are grouped together into myosins, kinesins, and dyneins—linear motor proteins that transport attached "cargoes" along microfilaments or microtubules (Figure 131).

Class="center">Table 6 - Molecular motors: energy source, Structure, and components

In Muscle tissue or eukaryotic flagella, the globular head of a linear (translational) motor protein uses the energy of ATP hydrolysis to slide an Actin filament relative to a microtubule (Figure 131(a)).

DNA and RNA polymerases also function as linear (translational) motors, as they translocate along DNA during Replication and transcription.

Rotary motors generate rotational movement responsible for flagellar rotation, DNA packaging into viral capsids, and ATP synthesis.

Table 7 - Molecular motors: intracellular localization and generated movement

The driving force for bacterial motility is generated by the rotational movement of a protein complex embedded in the bacterial membrane. Ions move down their electrochemical gradient through a stationary ring of proteins called the stator, which is anchored in the membrane. The torque generated by the stator drives the inner protein ring and the attached flagellum (Figure 131(6)).

Mitochondrial, thylakoid, and bacterial ATP synthases (F₀F₁ complexes) share a similar architecture.

Figure 131 - Operating principle of molecular motors: (a) linear motor; (b) rotary motor

6.7.1. ATP Synthase. ATP synthase is a combination of two motors, designated F₀ and F₁, which utilize different Energy Sources to drive mechanical rotation (Figure 132).

The membrane-embedded F₀ motor harnesses an electrochemical gradient, whereas the cytosolic F₁ motor operates via the energy of ATP hydrolysis.

In ATP synthase, these two motors are coupled such that one Functions as a motor and the other as a generator.

The rotation of the F₀ motor driven by proton translocation across the membrane can be used to synthesize ATP within the F₁ subunit. Conversely, the rotation of the F₁ motor powered by ATP hydrolysis can be used to pump protons across the membrane, thereby generating a proton gradient.

The F₀ motor consists of a cylindrical rotor and a stator comprising 10–14 identical subunits (depending on the Organism) fixed within the membrane. The membrane provides the barrier against which the proton gradient is established.

The cylindrical rotor terminates at its upper end in an eccentric cam that extends between the catalytic domains of the F₁ motor.

Figure 132 - Schematic representation of ATP synthase. The right panel shows a top view of the catalytic cytosolic portion of the F₁ motor and the membrane domains of the F₀ motor

As the rotor turns, the eccentric cam sequentially and mechanically deforms (stimulates) the catalytic subunits of the F₁ ATP motor, activating them to catalyze the attachment of a phosphate group to ADP.

The Mechanism of ATP synthase will be discussed in detail below (see Section 8.4.2).

All protein motors share three common properties:

1) The ability to convert the energy of a source—either ATP or an ion gradient—into translational or rotational movement,

2) the ability to bind and move along cytoskeletal filaments, nucleic acid strands, or Protein Assemblies,

3) coordinated movement in the desired direction.

6.7.2. Linear Motors. Motor proteins that move along cytoskeletal filaments can transport "cargo," which in the case of muscle cells and eukaryotic flagella are microtubules or B-tubules, respectively. These same motor Proteins can also transport chromosomes and vesicles along cytoskeletal filaments (Figure 133).

The two most thoroughly studied motor proteins are Myosin, which moves along actin filaments, and kinesin, which moves along microtubules.

Figure 133 - Cargo transport by kinesin

The globular heads of myosin and kinesin bind to cytoskeletal filaments, while the fibrillary "tail" attaches to the cargo—in our case, a vesicle. ATP hydrolysis within the globular heads drives their "walking" motion along the filament through repeated cycles of conformational changes.

These two motor proteins employ distinct principles for generating mechanical movement.

Each myosin molecule executes a single "power stroke" at a time—it binds to actin, pulls (or rather jerks) it, and then detaches from the actin.

In contrast, after attaching to a microtubule, kinesin performs multiple translocation steps, detaching from the microtubule only at the end of its "task."

These functional differences are tailored to various biological operations.

Myosin molecules are components of thick (massive) myosin filaments (complexes) that move along actin filaments—each myosin performs its "power stroke" and steps aside to make room for neighboring myosins to work.

Kinesin operates much more autonomously. It serves as a "delivery truck" for transporting cargo along a microtubule, requiring it to bind to the tubule and travel continuously along it.

Despite these functional differences, myosin and kinesin share a striking similarity in their molecular Organization, which is utilized to generate the "driving force."

All myosins share a similar architecture. Let us examine The structure of myosin II, which drives Muscle contraction (Figure 134).

6.7.3. Myosin. Myosin is composed of one or two heavy chains and several light chains.

The heavy chains consist of Three types of domains: a head, a neck, and a tail. The two globular head domains function as specialized ATPases that convert the energy of ATP hydrolysis into movement. Notably, the ATPase activity of the heads reaches its maximum when they are bound to actin.

Figure 134 - Schematic diagram of myosin II

The head terminates in an alpha-helical neck, to which light chains are non-covalently bound (wrapped around the neck). These light chains serve two purposes: (1) they amplify small Conformational Changes in the head into large spatial displacements of the molecule, and (2) they regulate head activity. They provide rigidity to the neck and can function as a lever arm for the head.

The filamentous tail domains contain binding sites that determine the specific activity of a given myosin.

The motor globular domain of myosin (the head) is elongated, and the ATP-binding site is located near its center (Figure 135).

Figure 135 - Three Stages of the myosin working cycle

In its relaxed state, myosin is bent, whereas the conformational transition induced by ATP binding straightens the molecule.

The myosin head consists of several functional domains.

The large catalytic domain binds to both ATP and actin.

The ATP-induced conformational change of the catalytic domain triggers a transition in the converter domain, which is further amplified by the lever arm domain, displacing the tip of the lever by 10 nm. In different myosin forms, the ends of this lever are anchored to appropriate Supports to perform specific mechanical work.

The myosin working cycle consists of three stages (Figure 135).

In The First stage (initial state), myosin is bound to ATP and dissociated from actin (Figure 135(a)).

In the second stage, ATP is hydrolyzed to ADP and phosphate, inducing a conformation of the actin-binding interface that allows actin binding, while myosin adopts a bent conformation (Figure 135(b)).

In the Third Stage, phosphate dissociates, causing myosin to straighten, which enables the "power stroke" (Figure 135(c)).

The exchange of ADP for ATP leads to the dissociation of myosin from the actin filament and returns myosin to its initial state.

6.7.4. Kinesin. Kinesin employs a fundamentally different mechanism for movement (Figure 136). It utilizes an unusual order-disorder transition within a short segment of the protein chain known as the neck linker, which connects the globular motor domain to the fibrillar tail.

Driven by ATP hydrolysis, the neck linker undergoes a conformational transition from a disordered state to an ordered structure, thereby executing a "power stroke".

The kinesin motor domain features a specialized groove into which the neck linker docks and undocks, depending on the chemical "cue" associated with its temporary, reversible binding to the motor domain.

The "power stroke" occurs when the neck linker becomes ordered and docks into the groove, associating with the motor domain. Whatever "cargo" is attached to the neck linker is thus displaced by 8 nm per "power stroke".

The kinesin working cycle begins with an initial state in which one subunit (shown in white) is empty, while the other (shown in grey) is bound to ADP (Figure 136, top).

ATP binds to the empty subunit, triggering a conformational transition in the "white" subunit; the neck linker becomes ordered, "docks" into the groove, and tightly binds to the globular head. Simultaneously, the white subunit detaches the grey subunit from the microtubule, swinging it "forward in the direction of movement" (middle section of Figure 136), where the grey subunit rebinds to the microtubule. ADP then dissociates As a result of the grey subunit binding to the microtubule.

Figure 136 - Stages of the kinesin working cycle

The dissociation of ADP from the grey subunit and the release of phosphate from the white subunit drive the reverse transition of the white subunit's neck linker from the ordered to the disordered state, causing it to detach from the groove of the globular head and resetting the entire system to its initial state.

Sequential stepping cycles allow kinesin to move along the microtubule.

Both myosin and kinesin respond to the presence of the third phosphate group in the ATP molecule and utilize ATP hydrolysis to generate a force impulse via a conformational transition.

6.7.5. Sarcomeres. The tail domains of hundreds of myosin molecules intertwine to form thick myosin filaments. The contractile units (sarcomeres) of vertebrate muscle fibers consist of alternating thick myosin filaments and thin actin filaments (Figure 137). Actin filaments are anchored to Z-discs. A sarcomere is defined as the longitudinal unit delimited by two Z-discs.

Figure 137 - Muscle contraction

Muscle fiber contraction is driven by the longitudinal sliding of thick myosin and thin actin filaments past one another. Coordinated conformational changes of hundreds of heads "pull" the myosin and actin filaments relative to each other. Myosin heads "walk" step-by-step along the actin filaments.

As shown in Figure 135, myosin undergoes a series of Conformations during each step. Over the course of a cycle, myosin must sequentially adopt at least three conformational states: (1) the ATP-bound, actin-detached state; (2) the ADP-P_i-bound, actin-attached state; and (3) the state following the completion of the actin filament "pulling" stroke.