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

Myosin II Voet Fig 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 & 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 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 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 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 Key features: ATP reduces Myosin’s affinity for actin Myosin-ADP strongly binds to actin Actin binding to Myosin induces phosphate release

Voet Fig Myosin Cycle 1.ATP binding Actin dissociation 2. ATP hydrolysis Cocking of myosin head 3. Weak actin binding4. Pi release Strong acting binding 5. Power stroke 6. ADP release Actin 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 Relay Helix Converter domain (green) Lever Arm Nucleotide Converter domain & Lever Arm Actin binding cleft

Myosin versus Kinesin Valle, Science 2000

Conformational changes in Myosin Voet Fig 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 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 KinesinMyosin Structure Conformational changes Power stroke Step size Processivity

Comparison of Myosin & Kinesin KinesinMyosin Structuresmalllarge same core: ATPase domain, relay helix Conformational changescomparable movement in relay helix different effect on power stroke Power strokeupon ATP bindingupon Pi release Step size (per head)16nm (8nm net)10nm Processivityhighly 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 release Step size: 10 nm Power stroke induced by ATP binding Step size: 8 nm Myosin Kinesin

Processivity Valle, Science 2000 Kinesin & Myosin V: Highly processive Myosin II: unprocessive Net 37 nm Step size Net 8 nm Step size

Evolution of Motor Proteins Valle, Science 2000