motor proteins - introduction part 2 biochemistry 4000 dr. ute kothe
TRANSCRIPT
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
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