Mapping the Cycle

The Big Picture: Mapping Mutants to Conformations to Biochemical states


Modelling the Musketeers: explaining the mutant-suppressor relationships we observe at position Y494, W501 and G691 (with G740 waiting in the wings to portray D'Artagnan!). Here, we focus on the 494-501 pairing, modelling it as a 'lock' that stabilizes the 'Pi-holding' state until external energy (providing by binding of actin) provides the 'boost' required to 'climb the mountain'--overcome the energetic barrier between states.

Explanation Structural Model: two states of the motor involving residues 494 and 501 Kinetic Interpretation
A) In the wild type case, we observe residues 494 and 501 interacting with one another in state I (as is observed in all extant crystal structures to date). We hypothesize an alternate state, in which 494 and 501 interact not with each other, but instead with other components of the motor.

Paired state stability prevents progress through the cycle. Height of purple arrow indicates energetic barrier to progress

B) Mutating position 494 disrupts the interaction with residue 501 in state I. However, in state II and the transition state the 494-X interaction is disrupted, but the 501-Y interaction is not. Thus overall we postulate that the relative barrier preventing 'forward progress' is lessened, allowing Pi release in the absence of actin binding.

Breaking up the pairing of 494 and 501 destabilizes the state on the left, lowering the relative barrier to reaction progress (purple arrow)

C) Symmetrically, if we instead mutate residue 501, we disrupt state I but only partially harm state II, since the 494-X interaction remains intact.

As above

D) The double mutant restores balance. This arises because while the 494-501 direct interaction was already in ruins (figures B and C), whereas the double mutant completes the demise of state II. Thus balance is restored (admittedly by damaging states I, II and the reaction intermediate), restoring the local kinetics to a semblance of normal function.

Further damage to state II and the reaction intermediate restores the height of the barrier preventing progress from state I (height of purple arrow)

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Bruce Patterson