Molecular Motors I

1. Molecular Motors I Andy HowardIntroductory Biochemistry 25 November 2008 Biochemistry: Motors

2. Chemistry and movement • Most purposeful biological motion is effected through actions of molecular motors. It’s worthwhile to understand the biochemistry of these systems Biochemistry: Motors

3. Definition Microtubules and their partners Tubulin Structure Cilia & flagella Microtubules (concluded) Movement of organelles Dyneins & kinesins DNA helicases Muscle contraction: for next time! Bacterial flagella What we’ll discuss Biochemistry: Motors

4. What is a molecular motor? • A protein-based system that interconverts chemical energy and mechanical work Biochemistry: Motors

5. Microtubules • 30-nm structures composed of repeating units of a heterodimeric protein, tubulin • -tubulin: 55 kDa • -tubulin: 55 kDa also • Structure of microtubule itself: polymer in which the heterodimers wrap around in a staggered way to produce a tube Biochemistry: Motors

6. Tubulin structure •  and  are similar but not identical • Structure determined by electron diffraction, not X-ray diffraction • Some NMR structures available too • Two GTP binding sites per monomer • Heterodimer is stable if Ca2+ present Biochemistry: Motors

7. iClicker quiz question 1 • Why might you expect crystallization of tubulin to be difficult? • (a) It is too big to crystallize • (b) It is too small to crystallize • (c) Proteins that naturally form complex but non-crystalline 3-D structures are resistant to crystallization • (d) It is membrane-bound • (e) none of the above Biochemistry: Motors

8. Tubulin dimer • G&G Fig. 16.2 Biochemistry: Motors

9. Microtubule structure • Polar structure composed of / dimers • Dimers wrap around tube as they move • Asymmetric: growth at plus end Biochemistry: Motors

10. Treadmilling • Dimers added at plus end while others removed at minus end (GTP-dependent): that effectively moves the microtubule • Fig. 16.3 Biochemistry: Motors

11. Role in cytoskeleton • Microtubules have a role apart from their role in molecular motor operations: • They are responsible for much of the rigidity of the cytoskeleton • Cytoskeleton contains: • Microtubules (made from tubulin) • Intermediate fibers (7-12nm; made from keratins and other proteins) • Microfilaments (8nm diameter: made from actin) Biochemistry: Motors

12. Cytoskeletal components • Fig. 16.4 Biochemistry: Motors

13. Cilia and flagella • Both are microtubule-based structures used in movement • Cilia: • short, hairlike projections, found on many animal and lower-plant cells • beating motion moves cells or helps moved extracellular fluid over surface • Flagella • Longer, found singly or a few at a time • Propel cells through fluids Biochemistry: Motors

14. Axonemes • Bundle of microtubule fibers: • Two central microtubules • Nine pairs of joined microtubules • Often described as a 9+2 arrangement • Surrounded by plasma membrane that connects to the cell’s PM • If we remove the PM and add a lot of salt, the axoneme will release a protein called dynein Biochemistry: Motors

15. Axoneme structure • Inner pair connected by bridge • Outer nine pairs connected to each other and to inner pair • Fig. 16.5 Biochemistry: Motors

16. How cilia move • Each outer pair contains asmaller, static A tubule anda larger, dynamic B tubule • Dynein walks along B tubulewhile remaining attached toA tubule of a different pair • Crosslinks mean the axoneme bends • Dynein is a complex protein assembly: • ATPase activity in 2-3 dynein heavy chains • Smaller proteins attach at A-tubule end Biochemistry: Motors

17. Dynein movement • Fig. 16.6 Biochemistry: Motors

18. Inhibitors of microtubule polymerization • Vinblastine & vincristine are inhibitors: show antitumor activity by shutting down cell division • Colchicine inhibits microtubule polymerization: relieves gout, probably by slowing movement of white cells Biochemistry: Motors

19. Paclitaxel: a stimulator • Formerly called taxol • Stimulates microtubule polymerization • Antitumor activity • Stimulates search for other microtubule polymerization stimulants Biochemistry: Motors

20. iClicker question 2 2. How do you imagine paclitaxel might work? • (a) by producing frantic cell division • (b) by interfering with microtubule disassembly, preventing cell division • (c ) by causing changes in tertiary structures of  and  tubulin • (d) none of the above Biochemistry: Motors

21. Movement of organelles and vacuoles • Can be fast:2-5 µm s-1 • Hard to study • 1985: Kinesin isolated • 1987: Cytosolic dynein found Biochemistry: Motors

22. Cytosolic dynein • Mostly moves organelles & vesicles from (+) to (-), so it moves things toward the center of the cell • Heavy chain ~ 400kDa, plus smaller peptides (53-74 kDa) • Microtubule-activated ATPase activity Biochemistry: Motors

23. Kinesin • Mostly moves organelles from (-) to (+) • That has the effect of moving things outward • 360 kDa: 110 kDa heavy chains, also 65-70 kDa subunits (2 + 2?) • Head domain of heavy chain (38 kDa) binds ATP and microtubule: cooperative interactions between pairs of head domains in kinesin, causing conformational changes in a single tubulin subunit • 8 nm movements along long axis of microtubule Biochemistry: Motors

24. Kinesin motion depicted • Rolling movement involving two head domains at a time • Fig. 16.8(b) Biochemistry: Motors

25. Hand-over-hand kinesin model • Two head groups begin in contact • After ATP hydrolysis hindmost head passes forward head • ATP binds to new leading head • Pi dissociates from trailing head Biochemistry: Motors

26. DNA helicases • To replicate DNA we need to separate the strands • Efficient only if the helicase can travel along the duplex quickly • This kind of movement is called processive • E.coli BCD helicase can unwind 33kbp before it falls off • If we want to replicate DNA rapidly, we need processivity Biochemistry: Motors

27. Achieving processivity • Some helicases form rings that encircle 1 or both strands of the duplex • Others, like rep helicase, are homodimeric; move hand-over-hand along the DNA, like kinesin Biochemistry: Motors

28. Negative cooperativity • Rep is monomeric without DNA • Each monomer can bind either ss or dsDNA • BUT after one monomer binds DNA, the second subunit’s affinity drops 104-fold! Biochemistry: Motors

29. Muscle contraction • This is an obvious case of an energy-dependent biological motion system • Involves an interaction called the sliding filament model, in which myosin molecules slide past actin molecules • Many other proteins and structural components involved • We’ll discuss this in detail next Tuesday Biochemistry: Motors

30. Bacterial flagella • E.coli flagellum is 10 µm in length, 15 nm in diameter • ~6 filaments on surface of cell rotate counter-clockwise: that makes them bundle together and propel the cell through medium • Enabled by rotation of motor protein complexes in plasma membrane Biochemistry: Motors

31. Motor structure • >= 2 rings, ~25nm diameter (M & S) • Rod attaches those to the helical filament • Rings surrounded by array of membrane proteins • This one is driven by a proton gradient, not by ATP hydrolysis: [H+]out > [H+]in, so protons want to move in • If we let protons in, we can use the thermodynamic energy to drive movement • Requires 800-1200 protons per full rotation! Biochemistry: Motors

32. The shuttle • MotA & MotB form shuttling device • Proton movement drives rotation of flagellar motor • Fig. 16.26 Biochemistry: Motors

33. iClicker question 3 3. Compare the pH inside the cell to the pH outside. • (a) pHin < pHout • (b) pHin> pHout • (c ) pHin = pHout • (d) We don’t have enough information to answer this question. Biochemistry: Motors

34. Berg’s model • motB has protonexchanging sites • motA has half-channels—one half facing toward the inside of the cell, one facing out • When a motB site is protonated, the outside edges of motA can’t move past it • Center of motA can’t move past site when it’s empty • Those constraints cause coupling between proton translocation and rotation Biochemistry: Motors

35. Coupling described • Proton enters outside of motA and binds to an exchange site on motB • motA is linked to cell wall, so when it rotates, it puts the inside channel over the proton • Proton moves through inside channel into cell; then another proton travels up the outside channel to bind to the next exchange site • That pulls the complex to the left, leading to counterclockwise rotation of disc, rod, & helical filament Biochemistry: Motors

36. Coupling depicted • Fig. 16.27 Biochemistry: Motors

37. What if it got reversed? • If outside became alkaline, the flagellar filaments would rotate clockwise • That doesn’t work as well because it loosens the microtubule Biochemistry: Motors

38. Quantitation • M ring has about 100 motB exchange sites • 800-1200 protons for a full rotation of the filament • That enables ~ 100 rotations/sec Biochemistry: Motors