Motor Proteins - Introduction Part 2 Biochemistry 4000 Dr. Ute Kothe.

Motor Proteins- Introduction Part 2

Biochemistry 4000

Dr. Ute Kothe

Myosin II

Voet Fig. 35-62

• Muscle Myosin

• ATPase

• 2 heavy chains (230 kDa): N-terminal globular head + C-terminal long -helical tail

• 2 essential light chains (ELC)

• 2 regulatory light chains (RLC)

• the -helical tails of 2 heavy chains form a coiled-coil

Actin

Voet Fig. 35-67 & 35-68

• ATPase

• Monomer called G-actin: active site in deep cleft, release of g-phosphates triggers conformational change

• polymer called F-actin: double chain of subunits, head-to-tail orientation

• (-) end: nucleotide binding cleft

• (+) end: opposite end

Microfilament Treadmilling

Voet Fig. 35-80

• Upon polymerization, F-actin hydrolyzes ATP and releases Pi

• Actin-ADP has lower affinity for other actin subunits

Newly polymerized (+) end containing Actin-ATP more stable than (–) end containing Actin-ADP

under steady state, subunits added to (+) end move toward (-) end where they dissociate

Structure of Striated Muscle

Voet Fig. 35-57

• Thick filaments (purple): myosin II coiled-coil tails packed end to end

• Thin filament (gray): F-actin + other proteins (Tropomyosin, Troponin)

• Protein-built Z-disk and M-disk which organize and anchor the thick and thin filament

Sliding Filament Model

Voet Fig. 35-70

• Lenght of thin and thick filaments remains constant

• Thick and thin filaments slide past each other

• Sliding is driven by many myosin heads (thick filament) walking along F-actin (thin filament)

• Overal results in contraction of muscle and generation of force

Myosin Cycle

Voet Fig. 35-71

Key features:

• ATP reduces Myosin’s affinity for actin

• Myosin-ADP strongly binds to actin

• Actin binding to Myosin induces phosphate release

Voet Fig. 35-73

Myosin Cycle1. ATP bindingActin dissociation

2. ATP hydrolysisCocking of myosin head

3. Weak actin binding4. Pi releaseStrong acting binding

5. Power stroke

6. ADP releaseActin is ADP release factor

Myosin cycle

Link to Movie on Bchm4000 webpage!

1. ATP binds to Myosin and induces opening of actin binding cleft; Myosin dissociates from actin.

2. ATPase catalytic site closes and ATP is hydrolyzed inducing a conformational change into high-energy state (cocking); myosin head is moved forward & perpendicular to F-actin

3. Myosin head binds weakly to actin one monomer further towards Z-disk

4. Myosin releases phosphate causing the actin binding cleft to close; strengthens myosin-actin interaction

5. Immediate power stroke: conformational change that sweeps myosin’s C-terminal tail about 10 nm toward the Z-disk relative to the motor domain (head)

6. ADP is released; actin acts as a nucleotide exchange factor

Myosin II Structure

Voet Fig. 35-62

Relay Helix

Converter domain(green)

Lever Arm

Nucleotide

Converter domain& Lever Arm

Actin bindingcleft

Myosin versus Kinesin

Valle, Science 2000

Conformational changes in Myosin

Voet Fig. 35-74

• Presence or absence of g-phosphates influences position of relay helix

• Changes in relay helix are transferred to converter domain

• Ultimately, result in large displacement of stiff lever arm

Is Myosin II a processive motor?

• the two myosin heads are not coordinate, cycle independently of each other

• net muscle contraction results from uncoordinated actin-attachement and -detachement of many myosins

Myosin II is not processive on its own!

Unconventional Myosin

Voet Fig. 35-86

• found in nonmuscle cells: often homodimers, some monomers

• mostly move to (+) end of actin, but Myosin VI travels to (-) end

• Myosin V: transports cargo via hand-over-hand mechanism, highly processive motor, large step size of net 37 nm per ATP hydrolysis (74 nm movement of one head)

Comparison of Myosin & Kinesin

Kinesin Myosin

Structure

Conformational changes

Power stroke

Step size

Processivity

Comparison of Myosin & Kinesin

Kinesin Myosin

Structure small large

same core: ATPase domain, relay helix

Conformational changes comparable movement in relay helix

different effect on power stroke

Power stroke upon ATP binding upon Pi release

Step size (per head) 16nm (8nm net) 10nm

Processivity highly non-processive

Myosin versus Kinesin

Valle, Science 2000

Similar structural elements (ATPase domain – blue, relay helix – green, mechanical elements – yellow)

Similar conformational changes in Motor domain

Power stroke in “different directions”

Red/light green:ADP/Nucleotide free

Yellow, dark green: ADP-Pi

Model for Power Strokes

Valle, Science 2000

Power stroke induced by Pi releaseStep size: 10 nm

Power stroke induced by ATP bindingStep size: 8 nm

Myosin Kinesin

Processivity

Valle, Science 2000

Kinesin & Myosin V:Highly processive

Myosin II:unprocessive

Net 37 nmStep size Net 8 nm

Step size

Evolution of Motor Proteins

Valle, Science 2000