Introduction to Molecular Motor Proteins: Part 1 Lecture Notes
Key Words and Terms
Molecular motors, kinesin, myosin, dynein, microtubules, actin, filament polarity, cytoskeleton, processivity, power stroke, ATPase cycle, x-ray crystallography, electron microscopy, in vitro motility assays
There are three classes of cytoskelelal motor proteins: kinesins, dyneins, myosins
Kinesin and dynein move along microtubules
Myosin moves along actin
There are other types of motors that are not discussed in this lecture: for example linear motors that move along DNA (helicases and polymerases) and rotary motors, such as the F1-Fo ATPase that produces ATP in mitochondria and the bacterial flagellar rotary motor.
Motor proteins are enzymes.
These enzymes hydrolyze ATP to generate phosphate and ADP, which are released sequentially, allowing the enzyme to rebind ATP and start another cycle.
During one cycle of ATP hydrolysis/product release, the motor undergoes a conformational change that can produce force and unidirectional motion. Kinesin, for example, takes an 8 nm step (the distance between adjacent a/b tubulin dimers on the microtubule) for each ATPase cycle. Cytoskeletal motor can work at 60% efficiency (efficiency is the theoretical chemical energy released from ATP hydrolysis that gets converted into work). This is much better than a car!
Cytoskeletal motor proteins have two major types of domains:
The Motor domain binds ATP and the cytoskeletal track. It is the “engine” that produces movement. The motor domain can be separated from the rest of the motor protein (by proteolysis, or by genetic engineering and expression) and it will produce movement in vitro (ie. in a test tube situation)
The Tail domain is everything in the polypeptide other than the motor domain. Part of the tail domain is involved in “cargo binding”, i.e. attaching the motor to the correct object in the cell that needs to be transported. Other segments might be involved in regulating (turning on or off) the motor activity. Yet other parts of the tail can be involved in dimerization of two motor protein polypeptide chains (e.g. through a coiled coil interaction) or higher order structures (e.g. muscle myosin also will polymerize into filaments).
Polymer tracks for cytoskeletal motors- actin and microtubules
Composed of a/b tubulin heterodimer (different isoforms exist)
A cylindrical, hollow polymer of 25 nm diameter
Very stiff , comparable to plexiglass
Composed of one protein- actin (although different isoforms exist)
A helical polymer with an 8 nm diameter
Much more flexible than a microtubule
Discussed more in the lecture by Julie Theriot on iBioSeminars
Actin and microtubule polymers are polar. The polarity is due to the fact that the individual subunits are asymmetric and they polymerize in a head-to-tail manner.
Motors recognize the polarity of the filament and travel in only one direction.
Kinesins travel towards the microtubule plus end
(Although one class of kinesin (Kinesin 13) travels to the minus end)
Dyneins travel towards the microtubule minus end
Myosins travel towards the actin plus end (also called “barbed end”)
(Although the Myosin VI class moves towards the plus end)
Cells organize the location and polarity of cytoskeletal filaments
In most cells, actin is found at the periphery, nucleated near the membrane. The plus ends point towards the membrane. In muscle, actin is very well ordered, with the plus end at the muscle Z line (not discussed in this lecture but found in many textbooks).
In most animal cells, microtubules are nucleated from the “centrosome” which is positioned near the nucleus (exceptions exist). The microtubules are long and can extend to cell periphery. The plus ends are located near the periphery and the minus ends are anchored at or near the centrosome. In a mitotic spindle, the microtubules’ plus ends interact with the chromosomes.
The defined polarity of filament organization and the fact that motors recognize this polarity creates a “navigation” system in the cell. For example, imagine that you are a small vesicle in the middle of the cell and your destiny is to travel to and fuse with the plasma membrane. You cannot “see” the plasma membrane and embark on that journey, as humans transport themselves to a destination. But you might have instructions to bind a kinesin motor to your surface. Then you (the vesicle) hop on the nearest microtubule track and kinesin will recognize the MT polarity and transport you to the near the plasma membrane. Once you get close, you can get to the plasma membrane by diffusion or pick up a myosin that will transport you through the actin network.
Motor proteins execute many types of biological activities
The lecture highlighted several types of activities for kinesins (a partial list)
Powering organelle transport
Positioning/transport of large organelles (e.g. Golgi, ER, nucleus)
Transport/localization of mRNAs
Ciliary biogenesis (movement of protein building blocks into cilia/flagella)
This same list largely pertains to dyneins and myosins. Dyneins also power the movement of cilia and flagella. Myosins are not involved in ciliary biogenesis but power muscle contraction (they are not involved in chromosome motion but pinch the dividing cell into two during the process of cytokinesis).
Motor proteins constitute “superfamilies”, and the different members are specialized for different biological tasks.
Every organism has different numbers of kinesins, myosins and dyneins
There are 45 Kinesin genes in human
They can be recognized by their similar “motor” domains (~30% identical in amino acid sequence)
Each gene has a very different “tail” domain (virtually no identity)- this allows for different localization, different cargo binding, unique regulatory control.
A similar situation is true for myosin and dynein superfamily members
How do molecular motors work?
The myosin motility cycle
Myosin binds ATP, hydrolyses the b-g phosphate bond, releases phosphate, and then releases ADP. ATP can rebind to restart the cycle.
These chemical transitions produce conformational changes as seen in the movie.
Many myosins simultaneously producing 10 nm strokes cause sarcomeres to shorten and your muscle to contract. Muscle myosin is not processive- it takes a “stroke” and then detaches from actin for most of its ATPase cycle. This is good for contracting muscle, since attached myosins finished with their “stroke” are not producing a “drag” for other stroking myosins (Other myosins in the cell, such as myosin V, are processive; see below).
The kinesin motility cycle
Kinesin is a processive motor. A single motor can move along a microtubule track for a hundred or more ATPase cycles without detaching. The two heads of kinesin move in a hand-over-hand manner, somewhat like walking across evenly spaced stepping stones placed across a pond.
These chemical transitions produce conformational changes as seen in the movie.
How do you study the mechanism of a molecular motor?
This provides atomic detail; you know how every amino acid in the protein is positioned in space and this gives one insight into the ATPase cycle and the conformational change mechanism.
Looking at 3-D atomic structures derived from this technique revealed a surprise- kinesin and myosin and even G proteins have similar structural features, indicating that they all evolved from a common ancestral protein. The feature that they most closely share is how they hydrolyze nucleotide and how they switch conformation after hydrolysis and phosphate release (ie between ATP and ADP states for motor proteins and GTP and GDP states for G proteins).
Even though kinesin and myosin are rather similar around their nucleotide binding regions, they have two major differences-
Electron microscopy (see techniques lecture by Eva Nogales)
No one has been able to obtain an X-ray crystal structure of a motor bound to its filamentous track. However, electron microscopy allows one to examine motor-track interactions, which is very informative, albeit at somewhat lower resolution than X-ray crystallography.
In vitro motility assays
These powerful methods allow one to study the dynamics of motor protein movement (EM and X-ray crystallography provide static views). These in vitro motility assays involve a purified motor, purified cytoskeletal filaments (ie polymerized from purified tubulin or actin), and ATP (which one can buy).
This lecture shows two motility assays-
New types of in vitro motility assays allow one to study single motor proteins
Using a method called an optical trap (see lecture 2 by Vale)- one can measure individual steps taken by motors (kinesin takes 8 nm steps, as shown by Steve Block and colleagues) and measure very small forces produced by motors (few piconewtons).
Other microscopes (using a technique called total internal reflection illumination and very sensitive modern cameras) allow one to visualize the fluorescence emitted from a single dye molecule. By putting a fluorescent dye or the green fluorescent protein onto a motor protein like kinesin, one can follow a single motor protein moving along its track.
Genetic engineering of motors proteins allows one to test theories of how they work.
Using expression plasmids, one can express and purify motile kinesin from bacteria. (Myosin is generally made in insect cells by baculovirus expression, and dynein has been expressed in yeast and Dictyostelium). Thus, one is not restricted to purifying motors from native tissues or cells, where their abundance might be very low. With expression systems, one also can change the sequence of the motor gene to test ideas of how the motor works and what parts of the protein contribute to its motor activity.
Motors and medicine
The lecture ends by describing the relevance of motor proteins to medicine, a big topic that is only briefly covered. Several human hereditary diseases are caused by mutations in motor protein genes. Small molecules directed against motor proteins might also have therapeutic benefit. Described in this lecture is a small molecule that activates cardiac myosin and improves cardiac contractility. This drug is being tested in humans in phase II clinical trials for patients suffering from heart failure (with impaired ventricular contraction). In section 3 of this lecture, a small molecular inhibitor of a mitotic kinesin is described.