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4 Molecular Motors and Machines

4 Molecular Motors and Machines

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11.4.1 Myosin

The myosins are a large family of molecular motors, where the human genome

includes about 40 myosin genes.

Myosin V is an actin-based molecular motor that has a key function in organelle

and messenger RNA (mRNA) transport, as well as in membrane trafficking. This

motor was the first member of the myosin superfamily shown to be processive,

meaning that a single motor protein can “walk” hand-over-hand along an actin

filament for many steps before detaching. The active state is extended whereas

the inactive state is compact (see [11.80]). Each myosin V molecule consists of

two heads (see Fig. 11.35) that contain an amino-terminal motor domain followed

by a lever-arm, a coiled-coil dimerization domain (S2), and a carboxy-terminal

globular cargo-binding domain. After ATP hydrolysis (see [11.15]), the loss of

the phosphate group from ATP leaves a space of approximately 0.5 nm, which is

thought to cause a rearrangement of structural elements flanking the ATP-binding

site. The rearrangement in this first level of amplification is coordinated with

structural changes in the actin-binding site. The next level of amplification involves

the communication of the initial conformational change in the active ATP site



Fig. 11.35 Orientation of the myosin V inhibited structure on actin from Spodoptera frugiperda

(Sf9) cells. (a) Only one myosin V head can be docked to the actin strand, rendered light-blue

and gray. The unbound head extends up to the viewer. (b) Cargo-binding domain (yellow) on the

motor domain (magenta) are rendered in a space-filling representation. The ATP-binding region

and Loop1 are colored green. A decrease in the flexibility of Loop1 may decrease ADP release rates

and the direct binding of the cargo binding domain to Loop 1 could stabilize its motion and decrease

ADP release rates, thus accounting for the decreased ATPase activity of the folded monomer.

(c) High-magnification electron micrographs of actin-bound myosin V molecules. (Reprinted with

permission from [11.80]. © 2006 Nature Publishing Group)



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to an α-helix mechanical amplifier which swings through an angle of up to 70◦ ,

presumably the ultimate cause for the working stroke and stepping of the motor

molecule. It is proposed that the myosin V motor after cargo delivery returns to its

starting position for rebinding cargo by an actin treadmilling and recycling process.

Myosin V cargo delivery would then be akin to delivering a package by “running

up the down escalator.” Once the cargo has been delivered to the actin end, myosin

V need only remain on the actin, which is facilitated by a conformation which

inhibits ATP turnover but not actin binding. The treadmilling process on actin will

eventually deliver myosin V to the other actin end where new cargo binding can

unfold and activate the molecular motor.

Myosin II motor molecules are the constituents of the thick filaments of muscle.

The α-helical coiled-coil myosin tails (see Fig. 11.34a) of the molecules form the

backbone of the thick filament (see Fig. 11.36a), whereas the two heads of each

molecule lie on the surface of the thick filament, where they can interact with the

actin thin filament subunits (see [11.15]). Muscles relax when this interaction is

blocked by molecular switches on either or both filaments. The structure of the

relaxed myosin II thick filament of tarantula spider muscles has been studied by

cryoelectron microscopy [11.81]. The repeating motif, occurring on the surface of

the filament at axial intervals of 14.5 nm (Fig. 11.36a), represent a pair of myosin

heads with a helical repeat of 43.5 nm. The filament backbone comprises 12 parallel strands, each ∼4 nm in diameter (and therefore containing more than one

2 nm diameter myosin tail), centered at a radius of ∼8 nm from the filament axis.

This study shows intramolecular interactions between the two motor heads of the

relaxed motor molecule (green and blue in Fig. 11.36b, c), between the motor heads

and the thick filament backbone, and intermolecular interactions between the motor

heads of adjacent molecules. These interactions may inhibit both the interaction of

the relaxed myosin II motor molecules with the actin filaments and their ATPase

activity. On activation of muscle, Ca2+ is released into the cytosol, increasing the

thick filament activity by phosphorylation of the regulatory light chains (RLC in

Fig. 11.36c). This breaks the bonds attaching the heads to each other and to the

thick filament surface, such that they become mobile and disordered. They now can

act independently of each other and are free to interact with the thin actin filament,

leading to contraction.

Skeletal muscles and their rapid contraction enables vertebrates to run, walk,

swim, or fly whereas involuntary movements such as heart pumping and gut peristalsis depend on the contraction of cardiac muscle and smooth muscles, respectively.

All these forms of muscle contraction depend on the ATP-driven sliding of highly

organized arrays of actin filaments against arrays of myosin II filaments (see

[11.15]). The long thin muscle fibers consist of huge single cells where the bulk of

the cytoplasm is made up of myofibrils, the basic contractile element with a diameter of ∼2 μm, which consists of tiny contractile units – called sarcomeres. Each

sarcomere [11.15] is formed from thin actin filaments, thick myosin II filaments,

and the spring-like titin proteins that give relaxed muscle its elasticity and which

can unfold its immunoglobulin-like domains one by one as stress is applied. Muscle

contraction can be activated by an incoming action potential which initiates Ca2+

flooding into the cytosol giving rise to the contraction of each sarcomere in the



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Fig. 11.36 Cryoelectron tomography of tarantula spider myosin II thick filaments. (a) Surface

view of the 3D tomography reconstruction with the repeating motif, representing a pair of myosin

heads with the shape of a tilted J. The filament backbone lying beneath the heads consists of subfilaments spaced ∼4 nm apart and running parallel to the filament axis. (b) Best fit of the atomic

structure of a relaxed pair of myosin II heads (space-filling model). (c) Ribbon presentation of the

atomic structure from (b) showing the motor domains (MD), the essential light chains (ELC), and

the regulatory light chains (RLC) of the blocked and the free heads, respectively. (Reprinted with

permission from [11.81]. © 2005 Nature Publishing Group)



cell. Each myosin II thick filament has about 300 heads and each head cycles about

five times/s in the course of rapid muscle contraction – sliding the myosin and the

actin filament past one another at rates of up to 15 μm/s with a sarcomere length

shortening by 10% in less than 20 milliseconds. Thus, muscle contraction consumes

enormous amounts of ATP by two processes: filament sliding, driven by the ATPase

of the myosin motor, and Ca2+ pumping, driven by the Ca2+ pump. The rapid synchronized shortening of the sarcomeres gives skeletal muscle the ability to contract

rapidly for running or flying, and even for playing the piano [11.15].



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11.4.2 Kinesin

Kinesin is a protein motor that ferries membrane-bound packages around cells. It

can carry a packet of neurotransmitter from the human spine to the tip of a finger

in about 2 days – a trip that would take a thousand years if left to simple diffusion

[11.82]. Conventional kinesin is a two-headed motor (see Fig. 11.34) that moves

along microtubules in the cytoskeletal scaffolding in 8 nm steps that match the

repeat distance of the microtubule structure. There is fair insight into the molecular mechanisms underlying this process: Each step requires the hydrolysis of one

molecule of adenosine triphosphate (ATP), the two kinesin heads move in a “handover-hand” fashion, and the movement stalls when the backward load exceeds ∼7

pN (see [11.82]). The head moves by unbinding in one hop within <50 μs [11.83].

By applying high loads to the kinesin it can be moved backwards, rather than

simply being ripped from the microtubule. This reversal of moving is similar to

that of a toy steam engine that can be made to run forward or backward by valve

changes [11.82, 11.83].

Individual kinesin motors can be tracked in living cells by the imaging of conjugated single quantum dots [11.84]. Streptavidin-conjugated semiconductor quantum

dots fluorescing at 655 nm were coupled to biotinylated conventional Drosophilae

kinesins (QD-K). These QD-Ks were internalized into the cytoplasm of cultured

mammalian HeLa cells (human epithelial cells) where the directed linear trajectories were observed in dependence of time (see Fig. 11.37) yielding a velocity of

v = 0.57 ± 0.02 μm/s similar to the in vitro motility [11.84].



Fig. 11.37 Tracking of a single quantum dot – kinesin conjugate in a part of a human epithelial

(HeLa) cell. (Reprinted with permission from [11.84]. © 2006 American Chemical Society)



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Some details of the kinesin-1 walk or of the myosin V stepping have been elucidated by fluorescence resonance energy transfer (FRET) experiments [11.85] or by

optical tweezer techniques [11.86], respectively.



11.4.3 Motor–Cargo Linkage and Regulation

Members of all three types of cytoskeletal motors are involved in organelle and

vesicle transport. To understand these functions, one has to determine how motors

link up to their cargoes and how transport is regulated [11.78]. In both processes,

non-motor domains and associated proteins have a key role for a wide spectrum

of attachment procedures (see Fig. 11.38). In some cell types, kinesin or dynein

can latch onto their cargo via membrane proteins. In neurons, the kinesin light

chains bind amyloid precursor protein (APP), a transmembrane protein of axonally transported vesicles. This link may be of medical significance as APP is the

precursor of the amyloid plaques in patients with Alzheimer’s disease (see Sect.

12.7). Impaired APP transport may well contribute to the development of the disease. The most wide-spread mode of association with cargo membrane proteins

occurs via linker proteins (see Fig. 11.38a, b). Among the myosins, the motor–

cargo linkage is characterized best. In pigment cells, the small GTPase Rab 27a

and a Rab-binding protein, melanophilin, attach myosin V to melanosomes (see

Fig. 11.38d and [11.15]).

From studies of motor–cargo association, the question of motor regulation in

cellular transport arises. Transport activity can be regulated either by turning the

motor on or off, or by inhibiting or promoting its association with cargo. In both

mechanisms, phosphorylation plays a significant role [11.79].



Fig. 11.38 Types of motor–cargo linkage. (a) Interaction between a transmembrane receptor and

kinesin heavy chains mediated by a linker protein (red). (b) Interaction between a transmembrane

receptor and kinesin light chains mediated by a linker complex (purple). (c) Interaction between

cytoplasmic dynein and an integral membrane protein mediated by the dynactin complex (red) and

spectrin (green). (d) Linkage of the tail domain of myosin V to membrane-anchored Rab 27a (red)

via melanophilin (purple). (Reprinted with permission from [11.79]. © 2003 Nature Publishing

Group)



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11.4.4 Diseases

A number of molecular motors are involved in diseases such as, e.g., in

myosin myopathies (myosin II), Griscelli syndrome (myosin V), hearing loss

(myosin IIIa, myosin II, etc.), the photoreceptor degeneration (retinitis pigmentosa;

cytoplasmic dynein), primary ciliary dyskinesia (axonemal dynein), polycystic kidney disease (dyneins and kinesins), Charcot–Marie tooth disease (Unc104 kinesin

family), anthrax susceptibility (Unc104 kinesin family), or neurodegenerative diseases (kinesin, cytoplasmic dynein) [11.79].



11.4.5 ATP Synthase (ATPase)

ATPase, nature’s smallest rotary motor, synthesizes ATP and, therefore, plays an

important roll in energizing the cell and the whole organism. In 1997 P. D. Boyer

and J. E. Walker were awarded the Nobel Prize in chemistry “. . . for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate

(ATP).” This protein is a rotor with a diameter of ∼10 nm and a length of ∼25 nm

with a shaft equipped with two parts – the enzymatic component F1 in the cytoplasm and a hydrophobic F0 component which is embedded in the membrane of the

mitochondria, the power plants of the cell (see Fig. 11.39). The negatively charged

F0 section is a biological nanoturbine driven by a proton current from the exterior



Fig. 11.39 Structure and functional principles of the ATPase enzyme. Left panel: a protein flux

(red arrows) through the F0 section gives rise to a rotation (yellow arrow) which is transferred

by the shaft (orange) to the F1 section where ATP synthesis occurs. Right panel: view of the F1

section from below. In the course of the synthesis cycle one of the three β subunits is empty (green),

one binds ADP and phosphate (not shown), and one contains the ATP reaction product. (Reprinted

with permission from [11.87]. © 2006 Wiley-VCH)



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to the interior of the cell (see [11.88]). The rotational motion of the ATPase motor

has been demonstrated by mounting an actin filament on the F1 section [11.89] from

which a torque τ of 2π τ = 3 G can be derived where G = 20 kB τ is the energy

difference between ATP and ADP. This indicates a high motor efficiency since three

ATP molecules are consumed per one rotation.

Each cell contains some 100 ATPase molecules and as the human body contains

some 1014 cells with each cell synthesizing 2 × 107 ATP molecules per hour the

body produces about 70 kg of ATP daily or even a ton under high strain.

The F0 component drives the rotation of the F1 component with the three

β subunits where ATP synthesis takes place (Fig. 11.39). One of the three β

binding pockets of F1 (see Fig. 11.39, right panel) is empty, one contains the starting molecule ADP, and one ATP as the synthesis product. The different binding

strengths of the ADP and the ATP molecules in the different β pockets originate

from the different conformations of these pockets which gives rise to ATP synthesis. The energy transferred by the rotating shaft initiates the release of the tightly

bound ATP molecule from the pocket.

The atomic processes occurring during ATP synthesis in the enzymatic F1 component when the ATPase shaft rotates have been studied by molecular dynamics

computer simulation [11.87, 11.90] taking into account 200,000 atoms. In this

“nanomechanical” process in the β subunits the tilting of the lower part of the subunit is transformed into a deformation of the ATP-binding pocket (Fig. 11.40). This



Fig. 11.40 Mechano-chemical energy conversion in the ATP-binding pocket of ATPase. Left

panel: a tilt movement (blue arrow) of the lower part of the β subunit (red and green) originating from the shaft rotation is transferred to the ATP-binding pocket (red arrows in the right panel)

giving rise to the release of the final ATP reaction product. Right panel: mechanically driven deformation of the binding pocket containing the ATP molecule (blue). (Reprinted with permission from

[11.87]. © 2005 Wiley-VCH)



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occurs by a sequence of structural changes where three positively charged amino

acids (arginines) play a role. The movement withdraws them from the negatively

charged ATP phosphate groups (see red arrows) which reduces the electrostatic

attraction so that the ATP molecule can be released from the binding pocket. This

behavior is similar to that of a combustion engine where the movements of the

β subunits corresponds to the piston movement and the rotation of the molecular

shaft to that of the crank shaft which synchronizes the mechano-chemical energy

conversion. In fact, the ATPase motor has been mechanically forced by external

electromagnets to rotate and generate chemical energy (ATP) [11.91].



11.5 Membrane Channels

Ion channels are nanometer-sized proteins with a seemingly simple task, but a highly

sophisticated machinery – to allow the passive flow of ions across biological membranes. The dimensions of these channels are some 5 nm long and fractions of a

nanometer wide. They must be highly selective for a particular type of ion, yet must

also maintain high transport rates and be able to regulate ion flow by switching it on

or off (“gating”). Ion channels are essential for important physiological processes

such as sensory transduction, action-potential generation, and muscle contraction, to

name just a few. When their function goes away, there can be serious consequences,

including life-threatening disease (see [11.92]). In 1998 the first crystal structure of

a bacterial potassium channel was revealed [11.93] with rapid progress hereafter, for

which R. MacKinnon won a Nobel chemistry prize in 2003. Here, we will give an

overview of the structures and dynamics of the K+ and the Ca2+ channels, the Cl−

channel, the characteristics of the aquaporin water channel, which was discovered

by P. Agre who shared the Nobel chemistry prize in 2003, and the main features of

protein channels.



11.5.1 The K+ Channel

Potassium (K+ ) channels are important membrane-spanning proteins that catalyze

the ionic movements required to generate electrical signals in nerve cells, where

the K+ concentration in the cell is about 30 times higher than outside. For structural studies of the K+ channel proteins, clearly diffracting crystals are required.

This could be helped by the use of a highly efficient system for expressing K+ ion

channels in bacteria [11.94] for large amounts of protein and by nibbling away the

channel proteins’ disorderly tops and tails – not essential to channel function – by

means of enzymes [11.93]. The x-ray structure of the K+ channel protein [11.93]

revealed the molecular design of these channels (Fig. 11.41a) with a high selectivity

and a rapid throughput that allows K+ to pass at high rates, while the channel simultaneously acts as a concrete wall to the smaller Na+ ion. This structure demonstrates

the molecular specificity (Fig. 11.41b, c): a narrow, 1.2 nm long, [11.96] “selectivity



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Fig. 11.41 Stereomolecular structure of the Streptomyces lividans bacterial K+ channel and mechanism of the K+ permeation. (a) A cutaway stereoview displaying the solvent-accessible surface

of the tetrameric K+ channel with the length of the selectivity filter (upper two green spheres)

of ∼1.2 nm, a total length of the channel of 4.5 nm, and the channel 1.0 nm wide in the middle

of the channel. The surface coloration varies smoothly from blue in areas of calculated high positive charge through white to red in negatively charged regions. The yellow areas of the surface

are colored according to carbon atoms of the hydrophobic side chains of protein residues in the

inner vestibule. The green spheres represent K+ ion positions in the conduction pathway [11.93].

(b) There are seven sites for K+ ions along the channel axis: one in the cavity site, four in the

selectivity filter, and two just beyond the external end of the channel. The cavity site is fully occupied, but (see c) only half of the remaining six are occupied at any one time. (c) The two main

ion configurations (outer and inner) that are postulated to exist within the channel. Purple arrows

indicate ion shifts that are linked directly to concerted ion entry into and exit from the pore with

red arrows indicating K+ shifts within the selectivity filter. Ion passage through the filter and extracellular sites occurs in a bucket-brigade fashion. (Reprinted with permission from [11.95]. © 2001

Nature Publishing Group)



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filter” in the shape of an oxygen-lined electronegative tunnel in which dehydrated

K+ (but not Na+ ) fits precisely. This structure rationalizes why a K+ ion is so willing

to leave the aqueous solution to enter the channel in a dehydrated form: the channel interior mimics the embrace of the water molecules in the hydration shell of

the ion in solution. Subsequent experiments [11.97, 11.98] and simulations [11.99]

showed how ions are coordinated in the filter; they reveal the K+ ion transit from

the channel into the aqueous solvent; and they indicate the character of water in

the cation’s inner hydration shell. In contrast to enzymes, channels process several substrate ions simultaneously in bucket-brigade fashion. The four filter sites

(Fig. 11.41c) are not all occupied simultaneously, but a pair of K+ ions separated

by a single water molecule shifts in a concerted fashion between two configurations

within the filter – inner and outer – occupying each about half the time (Fig. 11.41c).

When the K+ ion enters the channel from the solvent, it is coordinated in front by

four carbonyls reaching outward from the channel, and behind by solvent; this is

the “dehydration transition state” in which the ion sheds its water while entering the

pore (see [11.95]). Simulations [11.99] predicted the K+ ion right at the two sites

where it is seen by x-rays. The K+ -binding configurations and the transition between

them (Fig. 11.41c) are energetically similar, so that the entire conduction process is

barrier-less and very rapid.

In addition to the channel structure, the hydration shell of the aqueous cation

was determined in the channel’s central cavity containing ∼50 water molecules (see

[11.95]) with the oxygens of eight water molecules packed against the K+ ion at

the corners of a twisted cube. This closely matches the arrangement of the K+ coordinating oxygen in the selectivity filter which means that a flat energy landscape

is traversed by the K+ ions on their trip from one side of the membrane to the other.

Opening and closing mechanisms are necessary for most ion channels in addition

to selectivity in order to respond by conformational changes to metabolic cues. From

the structure of the Ca2+ -gated K+ channel of the Methanobacterium thermoautotrophicum [11.100] in the closed and in the open K+ -channel versions [11.100,

11.101] a model for gating is offered (see [11.96]). At the interface of the channel

with the cytoplasm inside the cell, protein bundles come together like an inverted

tepee, with eight domains forming the RCK (“regulates conduction of K+ ”) gating

ring which makes up the physical gate of the channel (see Fig. 11.42). This is just

located beneath the membrane cavity and the selectivity filter. In the crystal structure, two Ca2+ ions are clearly positioned at clefts between two RCK domains. The

Ca2+ binding presumably reshapes the clefts, expanding the diameter of the gating

ring and exerting a force on the attached channel-lining helices that opens the channel (see Fig. 11.42b). In this way the bacterial channel proteins use the chemical

Ca2+ binding energy to perform the mechanical work of gating.



11.5.2 The Ca2+ Channel

Communication between and within cells is essential for the development and

survival of any complex organism. Cells converse with each other through neurotransmitters and hormones that impinge on the cell surface, generating further



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Fig. 11.42 Model for the gating and opening of a bacterial Ca2+ gated K+ channel, based on

structural studies [11.100, 11.101]. (a) Closed conformation, (b) open conformation. Three of the

four subunits of the K+ channel are shown in brown. The purple and red circles represent the

eight RCK (“regulates conduction of K+ ”) domains, which, after binding Ca2+ , are thought to

reorientate with respect to each other, causing changes in the pore (opening) in the center of the

channel. (Reprinted with permission from [11.96]. © 2002 Nature Publishing Group)



signals (second messengers) within the cell which initiate appropriate responses.

The most ubiquitous of the second messengers is the calcium ion. An essential component for the regulation of the intracellular Ca2+ concentration has been

discovered by the forensic precision of molecular genetics [11.102, 11.104]. A

sharp rise in intracellular Ca2+ concentration can stimulate neurotransmitter release,

muscle contraction, cell metabolism, cell growth, or cell death. A primary route

for Ca2+ influx is the CRAC (for Ca2+ release-activated channel) which is activated by a fall in Ca2+ within the endoplasmic reticulum. Despite their biological

and clinical importance, very little about these channels, let alone their molecular composition, are known. Recently the protein Orai1 (encoded by the human

gene FLJ 14466) has been identified as being fundamental to the activation of

the Ca2+ influx channel [11.102, 11.103], although it is not yet clear whether this

could be the elusive CRAC channel. The possible roles of Orai1 in the CRAC Ca2+

channel mechanism, either being the entire channel, an indispensable subunit of

a multimeric channel complex, or a key component of the activation mechanism,

are depicted in Fig. 11.43. It is not even clear, whether the CRAC complex is

a channel through which ions would flow passively when it is open, or a transporter that uses energy to move ions across the membrane. In any case, the recent

progress will improve the prospects for developing therapeutic agents aimed at combating the growing list of human diseases associated with aberrant store-operated

calcium influx [11.105].



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