Kinesin: How it Waits Between Steps

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Kinesin How it Waits Between Steps
Holly Durst
Harvard Biovisions The Inner Life of the Cell
http//multimedia.mcb.harvard.edu/
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What is Kinesin?

• dimeric motor protein
• carries cellular cargo along microtubules by
  hydrolyzing ATP
• takes several hundred steps along a microtubule
  without detaching

Harvard Biovisions The Inner Life of the Cell
http//multimedia.mcb.harvard.edu/
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• ATP binding to leading head initiates neck linker
  docking and the other head is thrown forward
• New leading head docks onto binding site after
  diffusional search, resulting in 80 Å movement of
  attached cargo
• This accelerates ADP release and trailing head
  hydrolyzes ATP to ADP-Pi
• ATP binds to leading head

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FRET
http//bio.physics.uiuc.edu/images/FRET_concept.jp
g
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How kinesin waits between steps
Teppei Mori, Ronald D. Vale Michio Tomishige

• Objective
• Use a series of smFRET experiments to detect
  whether kinesin is bound to its microtubule track
  by one or two heads in its waiting conformation
  between steps

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The Players

• cysteine light human ubiquitous kinesin-1 dimer
  which cysteine residues and/or mutations were
  introduced into
• Dye-labelled kinesins were imaged moving along
  sea-urchin axonemes with a custom-built
  prism-type laser-illuminated total-internal
  reflection fluorescence microscope

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The Players
Used two FRET Sensors

• Heterodimer
• one chain containing single cysteine residue in
  plus end oriented tip of core (residue 215)
• one chain containing single cysteine residue in
  minus-end oriented base of the core (residue 43)

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The Players

• 2. Homodimer with a cysteine residue in both
  chains at the beginning of the neck linker
  (residue 324)

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Testing the Sensors

• Donor dye Maleimide modified Cy3
• Acceptor dye Maleimide modified Cy5
• Molecules that contained both dyes were selected
  for smFRET observations

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Testing the Sensors

• Examined FRET efficiency in presence of
  non-hydrolyzable nucleotide analog AMP-PNP so
  that both kinesin heads are bound statically to
  the microtubule

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Testing the Sensors
smFRET Efficiencies for 215-43
smFRET Efficiencies for 324-324

• Bimodal distribution of low (10) and high (90)
• As expected if two kinesin heads are bound to
  adjacent tubulin subunits 8 nm apart
• High peak 43 dye on leading head and 215 dye on
  trailing head
• Low peak 215 dye on leading head and 43 dye on
  trailing head

• Unimodal distribution centered at about 35
• Is consistent with a two-head bound state

Binding along single protofilament supported by
experiments with a 149-324 sensor
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Different Conditions
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Different Conditions

• Low ADP concentrations
• Remember ADP occupying binding site weak
  microtubule binding

215-43 Sensor unimodal at about 30

• 324-324 Sensor
• shift from 35 to 60
• kinesin heads come closer together

Under these conditions, these distributions
reflect a one-head bound state
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Different Conditions

• Low ADP plus excess inorganic phosphate

• Partial occupancy of an ADPPi state in tethered
  head

324-324 Sensor
215-43 Sensor

• Peaks characteristic of a two-head bound state
• Similar results for addition of AlF4-

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Different Conditions

• Both heads nucleotide free

215-43 Sensor
324-324 Sensor

• distributions similar to AMP-PNP, but with
  broader distributions
• Nucleotide-free kinesin primarily adopts a
  two-head bound state with partial occupancy of a
  one-head-bound state

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• FRET efficiency trace of individual
  axoneme-bound 215-43 heterodimer kinesin

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• Signal of 215-43 in AMP-PNP was fairly constant
• A subset of molecules with ADP or ADP/Pi or under
  nucleotide-free conditions underwent abrupt FRET
  transitions
• Unbinding and rebinding of kinesin head with
  microtubule

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Mutations

• Mutated so only one head could bind to
  microtubules under all conditions
• Y274A/R278A/K281A in loop 12 (L12-triple)
• 215(WT) 43 (L12)
• 215(L12) 43 (WT)

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Mutations
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Mutations

• 200 nM ADP
• 215(WT)-43(L12) and 215(L12)-43(WT) both produced
  unimodal distributions centered at about 30
• Distances between 43-labelled and 215 labelled
  dyes are similar
• Similar result for nucleotide-free state

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Mutations

• Addition of AMP-PNP
• 215(WT)-43(L12) bimodal with primary peak at
  80
• 215(L12)-43(WT) major peak shifted in opposite
  direction toward lower efficiencies
• movement of L12 triple towards plus-end oriented
  tip of bound head

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Mutations

• 215/342 dyes on wild-type chain to probe neck
  linker conformations in the bound head

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Mutations
Translation of unbound head from rear position to
forward position is driven by nucleotide-dependent
docking of neck-linker
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Dynamic Measurements

• Saturating ATP concentration (1 mM)
• Can only measure an average

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Dynamic Measurements

• 215-43 showed broad distribution centered at
  about 50
• Average of bimodal 10, 90 FRET distribution of
  static two-head bound kinesin
• Different from 30 value of one-head bound
  kinesin

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Dynamic Measurements

• 324-324 unimodal distribution centered at about
  30
• Kinesin spends most of the time bound with two
  heads to the microtubule when moving at
  saturating ATP concentrations

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Dynamic Measurements

• Subsaturating ATP concentration (2 µM)

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Dynamic Measurements

• 215-43 shifted to about 30
• 324-324 shifted to about 60
• More similar to 200 nM ADP (one-head bound)
• Suggests that kinesin waits primarily as a
  one-head bound intermediate when ATP binding
  becomes the rate-limiting step in the ATPase
  cycle

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Dynamic Measurements

• Longer dwell times at low ATP concentration

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Dynamic Measurements

• Spent most time in a roughly 30 FRET state
  (one-head bound) with brief spikes towards higher
  (80) FRET values
• Higher values represent transient two-head bound
  intermediate state
• Transitions from 30 to lower FRET state should
  also occur
• Difficult to distinguish from noise

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Dynamic Measurements

• Dwell-time histogram best fitted by a convolution
  of two exponentials
• Two rate-limiting ATP binding events occur
  between the two high-FRET spikes

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Dynamic Measurements

• Mean dwell time (140 ms) is comparable to
  predicted dwell time
• Total number of spikes divided by displacement of
  these molecules yielded an average distance of
  about 17 nm per spike
• Close to double kinesin step size

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Dynamic Measurements

• A kinesin step at low ATP concentrations involves
  a short-lived, two-head bound state, which then
  undergoes a transition to a longer-lived,
  one-head bound state

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Summary

• At high ATP concentration (the rate-limiting step
  is the detachment of the trailing head triggered
  by ATP hydrolysis/phosphate release), kinesin
  moves quickly from one two-head bound state to
  the next
• At low ATP concentration (ATP binding to the
  leading head is rate-limiting), the trailing head
  releases its Pi and detaches from the
  microtubule, producing a long-lived one-head
  bound state

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Summary
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Discussion

• Kinesin waits as either a one-head bound or
  two-head bound intermediate, depending on ATP
  concentration and the rate-limiting step

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Discussion

• ATPase cycles in the two kinesin heads are
  coordinated during processive motion
• Gating model proposes that detached head waits in
  front of bound head and is in a conformation that
  prevents it from binding tubulin
• But, transient interactions with the microtubule
  are seen
• Additional mechanism must keep detached head from
  progressing through ATPase cycle until its
  partner binds ATP

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Discussion

• Detached head will not release ADP when it is
  interacting with rear tubulin-binding site
• ADP release could occur after the bound head
  binds ATP and docks the neck-linker, translating
  the detached head to a forward tubulin-binding
  site
• Results are supported by Guydosh and Block who
  showed that nucleotide dissociation occurs only
  when a head is in the forward position
• Position dependence controlled by conformation of
  neck-linker

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Future Work

• How does the conformation of the neck-linker
  affect transitions in the ATPase cycle?

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References

• Mori, T. Vale, R. D. Tomishige, M. Nature 2007,
  450, 750-754
• Vale, R. D. Milligan, R. A. Science 2000, 288,
  88-95

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QUESTIONS?