Biochemistry, 4th Edition P54 pptx

Kinesin 1 KHCa b N type N-terminal domain Motor domain Coiled coil domain C-terminal tail domain Light chain Kinesin 13 MCAK Conventional kinesin kinesin I Myosin V Cytoplasmic dynein M

Kinesin 1 (KHC)

(a)

(b)

N type

N-terminal domain

Motor domain

Coiled coil domain

C-terminal tail domain

Light chain

Kinesin 13 (MCAK)

Conventional kinesin (kinesin I)

Myosin V

Cytoplasmic dynein

M type

Kinesin 14 (Ncd)

C type

Myosin V

Dynein

Tail

Calmodulin

Light chain

Kinesin light chain

AAA+

Light chains

Light chain

FIGURE 16.14 (a) Domain structure of kinesins, myosin

V, and cytoplasmic dynein (b) Molecular models of

kinesin 1, myosin V, and cytoplasmic dynein (Adapted from Vale, R., 2003 The molecular motor toolbox for intracellular

transport Cell 112:467–480.)

domain is located in different places in the sequence, depending on the function

of the specific family

The first dyneins to be discovered were axonemal dyneins, which cause sliding of microtubules in cilia and flagella Cytoplasmic dyneins were first identified in

Caenorhabditis elegans, a nematode worm Cytoplasmic dynein consists of a dimer of

two heavy chains (500 kD) with several other tightly associated light chains (Figure 16.14) Each heavy chain contains a large motor domain (380 kD) encompassing six AAA domains (see Section 16.4) arranged as a hexamer A 10-nm stalk composed

of a coiled coil projects from the head, between the fourth and the fifth AAA do-mains The stalk is the microtubule-binding domain

Myosin V is a multimeric protein that consists of 16 polypeptide chains The structure is built around a dimer of heavy chains, each of which includes head, neck, and tail domains The heavy chain head domain is virtually

indistinguish-HUMAN BIOCHEMISTRY

Effectors of Microtubule Polymerization as Therapeutic Agents

Microtubules in eukaryotic cells are important for the

mainte-nance and modulation of cell shape and the disposition of

intra-cellular elements during the growth cycle and mitosis It may

thus come as no surprise that the inhibition of microtubule

polymerization can block many normal cellular processes The

alkaloid colchicine (see accompanying figure), a constituent of

the swollen, underground stems of the autumn crocus

(Colchi-cum autumnale) and meadow saffron, inhibits the polymerization

of tubulin into microtubules This effect blocks the mitotic cycle

of plants and animals Colchicine also inhibits cell motility and

intracellular transport of vesicles and organelles (which in turn

blocks secretory processes of cells) Colchicine has been used

for hundreds of years to alleviate some of the acute pain of gout

and rheumatism In gout, white cell lysosomes surround and

engulf small crystals of uric acid The subsequent rupture of the

lysosomes and the attendant lysis of the white cells initiate an

inflammatory response that causes intense pain The

mecha-nism of pain alleviation by colchicine is not known for certain,

but appears to involve inhibition of white cell movement in

tis-sues Interestingly, colchicine’s ability to inhibit mitosis has

given it an important role in the commercial development of

new varieties of agricultural and ornamental plants When

mito-sis is blocked by colchicine, the treated cells may be left with an

extra set of chromosomes Plants with extra sets of

chromo-somes are typically larger and more vigorous than normal

plants Flowers developed in this way may grow with double the

normal number of petals, and fruits may produce much larger

amounts of sugar

Another class of alkaloids, the vinca alkaloids from Vinca rosea,

the Madagascar periwinkle, can also bind to tubulin and inhibit

microtubule polymerization Vinblastine and vincristine are used

as potent agents for cancer chemotherapy because of their ability

to inhibit the growth of fast-growing tumor cells For reasons that

are not well understood, colchicine is not an effective

chemother-apeutic agent, although it appears to act similarly to the vinca

alkaloids in inhibiting tubulin polymerization

The antitumor drug taxol was originally isolated from the bark

of Taxus brevifolia, the Pacific yew tree Like vinblastine and

col-chicine, taxol inhibits cell replication by acting on microtubules

Unlike these other antimitotic drugs, however, taxol stimulates

microtubule polymerization and stabilizes microtubules The

re-markable success of taxol in the treatment of breast and ovarian

cancers stimulated research efforts to synthesize taxol directly

and to identify new antimitotic agents that, like taxol, stimulate

microtubule polymerization

NH

OH

OH O

O

O O

O

O O

O O

OH

O

H3C

CH3

H3C

O

H3C

O

O

O

O

C

CH3

CH3

O

O

CH3 NH

C

O

CH3

O

C

H3CO

H3CO

N

HO OH

R N

N

N

Taxol

Colchicine

Vinblastine: R = CH3 Vincristine: R = CHO

䊱 The structures of vinblastine, vincristine, colchicine, and taxol.

able from the head domain of myosin II from skeletal muscle (see Figure 16.5),

but the neck domain is three times longer than the myosin II neck helix and it

contains six repeats of a calmodulin-binding domain Myosin V is normally

associ-ated with an essential light chain (similar to that of myosin II), together with

sev-eral calmodulins Adjoining the neck is a 30-nm-long coiled-coil domain The tail

domain of myosin V also binds a light chain and is adapted to bind specific

or-ganelles and other cargoes

Dyneins Move Organelles in a Plus-to-Minus Direction; Kinesins,

in a Minus-to-Plus Direction—Mostly

The mechanisms of intracellular, microtubule-based transport of organelles and

vesi-cles were first elucidated in studies of axons, the long projections of neurons that

ex-tend geat distances from the body of the cell In these cells, it was found that

sub-cellular organelles and vesicles could travel at surprisingly fast rates—as great as 1000

to 2000 nm/sec—in either direction

Cytosolic dyneins specifically move organelles and vesicles from the plus end of a

microtubule to the minus end Thus, dyneins move vesicles and organelles from the

Nucleus

Mitochondrion Golgi apparatus

Synaptic terminal

Multivesicular body Microtubule

Lysosome Cell body

Rough endoplasmic reticulum

(a)

Vesicles

(b)

Kinesin

Organelle Vesicle

FIGURE 16.15 (a) Rapid axonal transport along

micro-tubules permits the exchange of material between the

synaptic terminal and the body of the nerve cell (b)

Vesi-cles, multivesicular bodies, and mitochondria are carried through the axon by this mechanism (Adapted from a

cell periphery toward the centrosome (or, in an axon, from the synaptic termini

to-ward the cell body) Most kinesins, on the other hand, assist the movement of

or-ganelles and vesicles from the minus end to the plus end of microtubules, resulting

in outward movement of organelles and vesicles (Figure 16.15) Certain unconven-tional kinesins move in the opposite direction, transporting cargo in the plus-to-mi-nus direction on microtubules These kinesins have their motor domain located at the C-terminus of the polypeptide (see Figure 16.14)

Cytoskeletal Motors Are Highly Processive

The motors that move organelles and other cellular cargo on microtubules and actin filaments must be processive, meaning that they must make many steps along their cellular journey without letting go of their filamentous highway Dyneins, nonskeletal myosins, and most kinesins are processive motors (Table 16.2) Motors

in all these classes can carry cargo over roughly similar distances (700 to 2100 nm) before dissociating.1The step size of kinesin 1 is smaller than those of myosin V and cytoplasmic dynein; thus, its overall processivity (the average number of steps made before dissociating) is necessarily higher Moving at rates of 600 to 1000 nm/sec, these motors can carry their cargoes for a second or more before dissociating from their filaments

ATP Binding and Hydrolysis Drive Hand-over-Hand Movement of Kinesin

Kinesin movement along microtubules is driven by the cycle of ATP binding and hy-drolysis The molecular details are similar in some ways to those of the skeletal mus-cle myosin–actin motor but are quite different in other ways Kinesins, like skeletal muscle myosin, contain switch 1 and switch 2 domains that open and close in re-sponse to ATP binding and hydrolysis Together these switches act as a “-phosphate

sensor,” which can detect the presence or absence of the -phosphate of an adenine

nucleotide in the active site The switch movements between the ATP-bound and the ADP-bound states thus induce conformation changes that are propagated through

a relay helix to a neck linker that rotates, in ways similar to skeletal muscle myosin

(Figure 16.16) Thus, just as in skeletal myosin, small movements of the -phosphate sensor

at the ATP site are translated into piston-like movement of a relay helix and then into rotations

of the neck linker that result in motor movement

Here the kinesin and myosin models diverge, however, because the dimer of kinesin heavy chains translates these ATP-induced conformation changes into a hand-over-hand movement of its motor domain heads along the microtubule fila-ment Ronald Vale and Ronald Milligan have likened this movement of kinesin heads along a microtubule to a judo expert throwing an opponent with a forward swing of the arm

A model of kinesin motor movement is shown in Figure 16.17 Kinesin heads in solution (that is, not attached to a microtubule) contain tightly bound ADP

Bind-1 Compare these distances with the dimensions of typical cells in Table 1.2.

Distance Processivity

(cytoplasmic)

TABLE 16.2 Processivity of Motor Proteins

ing of one head of a kinesin multimer to a microtubule causes dissociation of ADP

from that head ATP binds rapidly, triggering the neck linker to rotate or ratchet

forward, throwing the second head forward as well and bringing it near the next

binding site on the microtubule, 8 nm farther along the filament The trailing head

then hydrolyzes ATP and releases inorganic phosphate (but retains ADP), inducing

its neck linker to return to its original orientation relative to the head Exchange

of ADP for ATP on the forward head begins the cycle again The structure of the

kinesin–microtubule complex (Figure 16.18) shows the switch 2 helix of kinesin in

intimate contact with the microtubule at the junction of the - and -subunits of a

tubulin dimer

The Conformation Change That Leads to Movement Is Different

in Myosins and Dyneins

The movement of myosin motors on cytoskeletal actin filaments is presumed to be

similar to the myosin–actin interaction in skeletal muscle Clearly, however, the

dif-ferent structure of the dynein hexameric motor domain and its associated

coiled-coil stalk (see Figure 16.14) must represent a different motor mechanism

ATP-dependent conformation changes in the ring of AAA modules must be translated

Converter

Neck linker

FIGURE 16.16 Ribbon structures of the myosin and kinesin motor domains and the conformational changes

triggered by the relay helix The upper panels represent the motor domains of myosin and kinesin, respectively, in

the ATP- or ADP-P i –like state Similar structural elements in the catalytic cores of the two domains are shown in

blue, the relay helices are dark green, and the mechanical elements (neck linker for kinesin, lever arm domains for

myosin) are yellow The nucleotide is shown as a white space-filling model The similarity of the conformation

changes caused by the relay helix in going from the ATP/ADP-P i –bound state to the ADP-bound or

nucleotide-free state is shown in the lower panels In both cases, the mechanical elements of the protein shift their positions

in response to relay helix motion Note that the direction of mechanical element motion is nearly perpendicular to

the relay helix motion (Adapted from Vale, R D., and Milligan, R A., 2000 The way things move: Looking under the hood of

molecular motor proteins Science 288:88–95.)

Kinesin

ADP

ADP

ADP

ATP

ATP

ADP-Pi

ATP

1

2

3

4

FIGURE 16.17 A model for the motility cycle of kinesin The two heads of the kinesin dimer work together to

move processively along a microtubule Frame 1: Each

kinesin head is bound to the tubulin surface.The heads are connected to the coiled coil by “neck linker” segments

(orange and red) Frame 2: Conformation changes in the

neck linkers flip the trailing head by 160°, over and be-yond the leading head and toward the next tubulin

bind-ing site Frame 3: The new leadbind-ing head binds to a new

site on the tubulin surface (with ADP dissociation), com-pleting an 80 Å movement of the coiled coil and the kinesin’s cargo During this time, the trailing head hydro-lyzes ATP to ADP and P i Frame 4: ATP binds to the leading

head, and P i dissociates from the trailing head, completing the cycle (Adapted from Vale, R., and Milligan, R., 2000.The way things move: Looking under the hood of molecular motor proteins.

Science 288:88–95.)

into movements of the tip of the coiled-coil stalk along a microtubule A proposed mechanism for dynein movement (Figure 16.19) suggests that the events of ATP binding and hydrolysis and ADP and Pirelease at an AAA module swing a linker that joins the AAA domain and the dynein tail

16.4 How Do Molecular Motors Unwind DNA?

The ability of proteins to move in controlled ways along nucleic acid chains is im-portant to many biological processes For example, when DNA is to be replicated, the strands of the double helix must be unwound and separated to expose single-stranded DNA templates Similarly, histone octamers (Figure 11.26) slide along DNA strands in chromatin remodeling, Holliday junctions (see Figure 28.22) move, and nucleic acids move in and out of viral capsids The motor proteins that move directionally along nucleic acid strands and accomplish these many functions are

called translocases The translocases that unwind DNA or RNA duplex substrates are termed helicases Thus, all helicases are translocases, but not vice versa.

FIGURE 16.18In the kinesin–microtubule complex, the

switch 2 helix (yellow) of kinesin (left) lies in contact

with the microtubule (right) at the subunit interface of a

tubulin dimer (pdb id  2HXH).

(a)

Motor domain

Microtubule

(b)

Cargo, such as vesicles

ADP+ Pi

= ADP bound

= ATP bound

Stalk Head Linker Tail

ATP

FIGURE 16.19 A mechanism for the dynein power stroke involves conformation changes in the head domain

(a) that facilitate movement of the stalk along a microtubule (b) ATP binding to the motor domain promotes

dissociation of dynein from the microtubule Hydrolysis of ATP causes a conformation change that primes the structure for a power stroke Microtubule movement is initiated by tight binding to the tip of the stalk, which promotes a conformation change in the head ring (the power stroke) Release of ADP and P i from the catalytic site causes tilting of the stalk at the end of the cycle (Adapted from Oiwa, K., and Sakakibara, H., 2005 Recent progress

in dynein structure and mechanism Current Opinion in Cell Biology 17:98–103.)

All translocases and helicases are members of six protein “superfamilies” (Table

16.3 and Figure 16.20), all of them related evolutionarily to RecA, a DNA-binding

protein (pages 881–882) Motors of superfamily 1 (SF1) and superfamily 2 (SF2)

consist of two RecA domains in a tandem repeat Motors of SF3 through SF6 are

built from single RecA domain peptides that associate to form hexamers and

dode-camers Each superfamily possesses characteristic conserved residues and sequence

elements (Table 16.3), most of which are shared between several superfamilies All

members of a given superfamily move in the same direction on a DNA or RNA

tem-plate (either 5 to 3 or 3 to 5) The hexameric motor proteins of the SF3 and SF6

superfamilies are members of the ancient AAAⴙ ATPase family (AAA stands for

“ATPases associated with various cellular activities,” and the “” sign refers to an

ex-panded definition of the family characteristics.)

Translocases and helicases, like other molecular motors, require energy for their

function The energy for movement along a nucleic acid strand, as well as for

sepa-ration of the strands of a duplex (DNA or RNA), is provided by hydrolysis of ATP

Translocases and helicases move on nucleic acid strands at rates of a few base pairs

to several thousand base pairs per second These movements are carefully regulated

by accessory proteins in nearly all cases Translocases and helicases typically move

MCM

Zn binding

Superfamily 6 (MCM)

Origin-binding domain

Superfamily 5

Primase

Superfamily 4

Origin binding

Superfamily 3 (BPV E1)

Protein:protein?

Protease

Superfamily 2

DNA binding Unknown DNA binding

Core domains Accessory domains Superfamily 1

TABLE 16.3 Helicase Superfamilies

*A: helicase moves 3 →5 on nucleic acid B: helicase moves 5→3.

along the DNA or RNA lattice for long distances without dissociating This is termed

processive movement,and helicases are said to have a high processivity For example,

the E coli BCD helicase, which is involved in recombination processes, can unwind

33,000 base pairs before it dissociates from the DNA lattice Processive movement is es-sential for helicases involved in DNA replication, where millions of base pairs must be replicated rapidly

Helicases have evolved at least two structural and functional strategies for achiev-ing high processivity The hexameric helicases (of the SF3 through SF6 superfamilies) form ringlike structures that completely encircle at least one of the strands of a DNA

duplex The SF1 and SF2 helicases, notably Rep helicase from E coli, are monomeric

or homodimeric and move processively along the DNA helix by means of a “hand-over-hand” movement that is remarkably similar to that of kinesin’s movement along microtubules A key feature of hand-over-hand movement of a dimeric motor protein along a polymer is that at least one of the motor subunits must be bound to the poly-mer at any moment

Negative Cooperativity Facilitates Hand-over-Hand Movement

How does hand-over-hand movement of a motor protein along a polymer occur? Clues have come from the structures of Rep helicase and its complexes with DNA

The Rep helicase from E coli is a 76-kD protein that is monomeric in the absence

of DNA Binding of Rep helicase to either single-stranded or double-stranded DNA induces dimerization, and the Rep dimer is the active species in unwinding DNA Each subunit of the Rep dimer can bind either single-stranded (ss) or

double-stranded (ds) DNA However, the binding of Rep dimer subunits to DNA is negatively

co-operative (see Chapter 15) Once the first Rep subunit is bound, the affinity of DNA

for the second subunit is at least 10,000 times weaker than that for the first! This negative cooperativity provides an obvious advantage for hand-over-hand walking When one “hand” has bound the polymer substrate, the other “hand” releases A conformation change could then move the unbound “hand” one step farther along the polymer where it can bind again

But what would provide the energy for such a conformation change? ATP hydrolysis is the driving force for Rep helicase movement along DNA, and the neg-ative cooperativity of Rep binding to DNA is regulated by nucleotide binding In the absence of nucleotide, a Rep dimer is favored, in which only one subunit is bound

to ssDNA In Figure 16.21a, this state is represented as P2S [a Rep dimer (P2) bound

AMP-PNP

(a)

(b)

FIGURE 16.20 (a) Translocase and helicase motors of

SF1 and SF2 are monomers that consist of two RecA

domains in a tandem repeat (pdb id  1QHG).

(b) Motor peptides of SF3 through SF6 associate to

form hexamers (as shown) or dodecamers

(pdb id  1CR0).

(b)

1B

Translocation

ATP

Active unwinding

(a)

ADP + Pi

3 

3 

FIGURE 16.21 (a) A hand-over-hand model for movement along (and unwinding of ) DNA by

E coli Rep helicase The P2 S state consists of a Rep dimer bound to ssDNA The P 2 SD state

in-volves one Rep monomer bound to ssDNA and the other bound to dsDNA The P 2 S 2 state has

ssDNA bound to each Rep monomer ATP binding and hydrolysis control the interconversion of

these states and walking along the DNA substrate (b) Crystal structure of the E coli Rep helicase

monomer with bound ssDNA (dark blue, ball and stick) and ADP (red) The monomer consists of

four domains designated 1A (residues 1–84 and 196–276), 1B (residues 85–195), 2A (residues

277–373 and 543–670), and 2B (residues 374–542) The open (purple) and closed (green)

confor-mations of the 2B domain are superimposed in this figure (pdb id  1UAA) (From Korolev, S.,

Hsieh, J., Gauss, G., Lohman, T L., and Waksman, G., 1997 Major domain swiveling revealed by the crystal

struc-tures of complexes of E coli Rep helicase bound to single-stranded DNA and ADP Cell 90:635–647 Reprinted

to ssDNA (S)] Timothy Lohman and his colleagues at Washington University in

St Louis have shown that binding of ATP analogs induces formation of a complex

of the Rep dimer with both ssDNA and dsDNA, one to each Rep subunit (shown as

P2SD in Figure 16.21a) In their model, unwinding of the dsDNA and ATP

hydroly-sis occur at this point, leaving a P2S2state in which both Rep subunits are bound to

ssDNA Dissociation of ADP and Pileave the P2S state again (Figure 16.21a)

Work by Lohman and his colleagues has shown that coupling of ATP

hydroly-sis and hand-over-hand movement of Rep over the DNA involves the existence of

the Rep dimer in an asymmetric state A crystal structure of the Rep dimer in

com-plex with ssDNA and ADP shows that the two Rep monomers are in different

con-formations (Figure 16.21b) The two concon-formations differ by a 130° rotation about

a hinge region between two subdomains within the monomer subunit The

hand-over-hand walking of the Rep dimer along the DNA surface may involve

alterna-tion of each subunit between these two conformaalterna-tions, with coordinaalterna-tion of the

movements by nucleotide binding and hydrolysis

Papillomavirus E1 Helicase Moves along DNA on a Spiral Staircase

Papillomaviruses are tumor viruses that cause both cancerous and benign lesions in

a host Replication of papillomaviral DNA within a host cell requires the

multifunc-tional 605-residue viral E1 protein Monomers of E1 assemble at a replication

ori-gin on DNA and form a pair of hexamers that wrap around a single strand of DNA

These assemblies are helicases that operate bidirectionally in the replication of

viral DNA The N-terminal half of the E1 protein includes a regulatory domain and

a sequence-specific DNA-binding domain, whereas the helicase activity is located in

the C-terminal half of the protein The C-terminal helicase domain (Figure 16.22)

includes a segment involved in oligomer formation (residues 300 to 378) and an

AAA domain (residues 379 to 605)

AAA domains are found in proteins of many functions, including motor

activ-ity by dyneins (see Section 16.3) and helicases, protein degradation by proteasomes

(see Chapter 31), and disassembly of SNARE complexes (see Chapter 9) This

ubiq-uitous module consists of two subdomains: an N-terminal segment known as an

/ Rossman fold, and a C-terminal -helical domain (Figure 16.23) The Rossman

fold is wedge-shaped and has a -sheet of parallel strands in a 5-1-4-3-2

pat-tern Key features of this fold include a Lys residue in the Walker A motif, an

Asp–Asp or Asp–Glu pair in the Walker B motif, and a crucial Arg residue in a

struc-ture called an arginine finger These three motifs are essential for ATP binding and

(a)

100 Å

(b)

F

E

D

C

B

A

FIGURE 16.22 (a) The papillomavirus E1 protein is a

605-residue monomer that forms hexameric assemblies at specific sites on single-stranded DNA (pdb id  2V9P).

(b) The C-terminal helicase domain shown here

includes an oligomerization domain (magenta) and the AAA  domain (blue).

FIGURE 16.23The AAA  domain is composed of an N-terminal, wedge-shaped Rossman fold and a C-terminal -helical domain (upper left).The P-loop

(red), the Walker A motif (purple), the Walker B motif (yellow), and the arginine finger (blue) are shown The ATP-binding sites lie between subunits of the hexamer Each ATP site includes the arginine finger of one sub-unit and the Walker A and Walker B motifs of the adja-cent subunit (pdb id  1D2N).

P-loop

Walker A

Walker B

hydrolysis In an AAA hexamer, the ATP-binding sites lie at the interface between any two subunits, involving the arginine finger of any given subunit and the Walker

A and Walker B motifs of the adjacent subunit

The structure of a large fragment (residues 306 to 577) of the papillomavirus E1 protein bound to a segment of ssDNA (Figure 16.24) reveals the remarkable mech-anism by which this helicase traverses a DNA chain The oligomerization domains form a symmetric hexamer, but the six AAA domains each display a unique con-formation The DNA strand is bound in the center pore of the AAA hexamer, with six nucleotides of the DNA chain each bound to residues from each of the protein subunits The crucial nucleotide-binding residues include Lys506 and His507 on a hairpin loop and Phe464, all of which face the center pore of the protein (Figure 16.25) Lys506interacts with one DNA phosphate oxygen, and His507forms a hydro-gen bond with the phosphate of an adjacent nucleotide in the DNA chain The aliphatic portion of Lys506and the aromatic groups of Phe464and His507share van

der Waals interactions with the DNA sugar moiety linking these two phosphates The

F

E

D

C B

A

FIGURE 16.24A view of the E1 helicase along the

ssDNA axis, showing the DNA-binding hairpin loops

from each monomer (colored) interacting with the

phosphates of DNA DNA is shown as a ball-and-stick

model in the center of the structure (pdb id  2GXA).

FIGURE 16.25The hairpin loops of each subunit in the

E1 helicase interact with two adjacent nucleotides in

the DNA chain Interactions include an ionic bond

between Lys 506 (yellow) and a DNA phosphate oxygen, a

hydrogen bond between His 507 and the phosphate of

an adjacent nucleotide, and van der Waals interactions

between the aromatic rings of Phe 464 (purple) and His 507

(olive) and the aliphatic chain of Lys 506 , with the sugar

linking the two phosphates (pdb id  2GXA).