Case study D · Investigate the molecules

Molecular machinery: ATP synthase and kinesin

ATP synthase makes most of a cell's ATP by turning. A flow of protons spins a ring in the membrane, and the rotation squeezes ADP and phosphate together in three catalytic sites. Kinesin walks cargo along microtubules, one ATP per 8-nanometre step.

β subunits show the binding-change cycle: open, loose, tight. Schematic, not to scale.

Select a part to see what it does. In mitochondria the shaft turns about 100 times a second (Watt et al. 2010); isolated F₁ reaches about 130 revolutions per second (Yasuda et al. 2001). The animation is slowed so the cycle can be followed.

Kinesin: a walking motor

microtubule: tubulin dimers every 8 nmcargo
Steps (8 nm each)0
Distance0 nm
ATP used0
StatusWalking

Kinesin's two heads take turns, hand over hand (Yildiz et al. 2004). Each 8-nm step uses one ATP (Schnitzer & Block 1997), and a motor typically takes about 100 steps before it lets go. Here each step has a 1% chance of ending the run, so run lengths vary as they do in single-molecule experiments. Kinesin and myosin share a core motor structure (Kull et al. 1996).

Methodology and limitations

How this works, and what it leaves out

Method

  • The ATP synthase model follows the subunit arrangement in the crystal and cryo-EM structures (Abrahams et al. 1994; Stock et al. 1999; Watt et al. 2010). Protons per ATP are c / 3, since one turn moves c protons and makes 3 ATP.
  • The β subunits change colour with the angle of the central stalk to show Boyer's binding-change cycle. "Show 120° stepping" uses the 90° + 30° substeps measured in isolated F₁ (Yasuda et al. 2001).
  • Kinesin takes 8-nm steps with one ATP each; detachment is modelled as a constant 1% chance per step, which gives run lengths averaging about 100 steps.

Limitations

  • Both models are schematic and slowed down. Real stepping was measured in F₁ splitting ATP; in the whole enzyme making ATP the rotor turns the other way, driven by protons.
  • Proton-to-ATP ratios in living cells can differ from c/3 because of leaks and slip.
  • Kinesin's mechanochemistry (neck-linker docking, the two heads' coordination) is reduced to alternating steps.
Evidence and debate

Weighing the evidence

Addresses: The emergence of molecular machinesThe origin of life

01 What is observed?

02 What does evolutionary theory propose?

  • SpeculativeRotary ATPases began as membrane protein translocases, which in turn evolved from RNA translocases. In those ancestors the translocated polymer sat where the central stalk is now. (Mulkidjanian et al. 2007)
  • Active researchKinesins and myosins descend from a common ancestral nucleotide-binding motor and diverged to walk on different tracks. (Kull et al. 1996; Vale & Milligan 2000)
  • SpeculativeCoupling to ion gradients (chemiosmosis) may go back to natural proton gradients at alkaline hydrothermal vents. (Lane & Martin 2012)

03 What are the unresolved questions and criticisms?

  • Active researchNo stepwise route to ATP synthase in which each stage is shown to work has been reconstructed. Homology identifies related parts; it does not by itself show the path. (Mulkidjanian et al. 2007)
  • Active researchTurning proton flow into rotation needs the a subunit and the c-ring to work together. How that coupling arose in stages is an open question. (The Lab's summary.)
  • Active researchAlmost all cells use ATP synthase to make the ATP that their other machinery, including the machinery that builds ATP synthase, depends on. Its origin is therefore bound up with the origin of life. (Lane & Martin 2012)

04 What evidence would help distinguish competing explanations?

  • Resurrected ancestral rotary-ATPase subunits, tested for the partial functions proposed for them.
  • Simpler rotary systems in nature that work with fewer parts.
  • Laboratory experiments that couple an ion channel to a ring ATPase and select for rotation or transport.

Established Measured or observed repeatedly; not in serious dispute.Active research An open research question, or a claim that specialists still dispute.Speculative A proposed explanation that has not yet been tested directly.

Your conclusions

Before you move on

Your notes stay in this browser. Nothing is sent anywhere unless you choose to include them in a link you share.

References

  1. Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994). Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370:621–628. doi:10.1038/370621a0
  2. Boyer PD (1997). The ATP synthase: a splendid molecular machine. Annual Review of Biochemistry 66:717–749. doi:10.1146/annurev.biochem.66.1.717
  3. Ibuki T, Imada K, Minamino T, et al. (2011). Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases. Nature Structural & Molecular Biology 18:277–282. doi:10.1038/nsmb.1977
  4. Imada K, Minamino T, Tahara A, Namba K (2007). Structural similarity between the flagellar type III ATPase FliI and F1-ATPase subunits. PNAS 104:485–490. doi:10.1073/pnas.0608090104
  5. Kull FJ, Sablin EP, Lau R, Fletterick RJ, Vale RD (1996). Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature 380:550–555. doi:10.1038/380550a0
  6. Lane N, Martin WF (2012). The origin of membrane bioenergetics. Cell 151:1406–1416. doi:10.1016/j.cell.2012.11.050
  7. Mulkidjanian AY, Makarova KS, Galperin MY, Koonin EV (2007). Inventing the dynamo machine: the evolution of the F-type and V-type ATPases. Nature Reviews Microbiology 5:892–899. doi:10.1038/nrmicro1767
  8. Noji H, Yasuda R, Yoshida M, Kinosita K (1997). Direct observation of the rotation of F1-ATPase. Nature 386:299–302. doi:10.1038/386299a0
  9. Schnitzer MJ, Block SM (1997). Kinesin hydrolyses one ATP per 8-nm step. Nature 388:386–390. doi:10.1038/41111
  10. Stock D, Leslie AGW, Walker JE (1999). Molecular architecture of the rotary motor in ATP synthase. Science 286:1700–1705. doi:10.1126/science.286.5445.1700
  11. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993). Direct observation of kinesin stepping by optical trapping interferometry. Nature 365:721–727. doi:10.1038/365721a0
  12. Vale RD, Milligan RA (2000). The way things move: looking under the hood of molecular motor proteins. Science 288:88–95. doi:10.1126/science.288.5463.88
  13. Walker JE, Saraste M, Runswick MJ, Gay NJ (1982). Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. The EMBO Journal 1:945–951. doi:10.1002/j.1460-2075.1982.tb01276.x
  14. Watt IN, Montgomery MG, Runswick MJ, Leslie AGW, Walker JE (2010). Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. PNAS 107:16823–16827. doi:10.1073/pnas.1011099107
  15. Yasuda R, Noji H, Kinosita K, Yoshida M (1998). F1-ATPase is a highly efficient molecular motor that rotates with discrete 120° steps. Cell 93:1117–1124. doi:10.1016/s0092-8674(00)81456-7
  16. Yasuda R, Noji H, Yoshida M, Kinosita K, Itoh H (2001). Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410:898–904. doi:10.1038/35073513
  17. Yildiz A, Tomishige M, Vale RD, Selvin PR (2004). Kinesin walks hand-over-hand. Science 303:676–678. doi:10.1126/science.1093753