Toggle Switches: setting states of the motor

Parts of protein machines tend to move as units (domains) and in general exist in only one of two states. Which state a given domain adopts depends on its relative 'comfort level' in that state. The comfort level, in turn, is determined by the sum of all the favorable and unfavorable atomic interactions taking place within the domain and between the domain and itself, its neighbors, and its environment.

So what's the point of this preamble? Simply stated, by altering the amino acid components of a domain, we potentially modify the domain's relative comfort in its alternative positions. To return to our tired car engine analogy, if we made a valve bigger, it'd be much more comfortable in the 'open' position than the closed. The same general idea would hold for the myosin motor--instead of being a finely tuned machine with a progression of states, a well-chose mutant could cause it to favor one of 2 previously balanced states. This would impede the motor just as a sticking valve would.

The upshot of this is that we predict that there should be key points (amino acid residues) in the myosin motor that exist as finely tuned 'switches' in their natural (wild type) state. By mutating these to at least some of the other 19 amino acids, we will 'throw' the switch toward one of its alternatives. Indeed, in some cases we'll be able to identify BOTH alternatives--mutants that bias the domain to state A and other mutants that bias the domain toward its alternative, state B.

A fine example of this sort of thing can be found in our genetic characterization of the G680V mutation. As described elsewhere, this mutation appears to 'stick' the motor in a state where it has interacted productively with actin, but can't seem to 'spit out' the Pi molecule that it has chopped off of ATP. In other words, we've 'toggled' the motor to the 'sit before you spit' state. In our search for suppressors of this mutation, we discovered the L176F mutation. In combination with G680V, we again achieve a balanced, functional motor. On its own, L176F is a 'drooler'--it lets out the Pi molecule even without the 'permission' normally granted by productive actin binding. As you can see in the Amino Acid Theatre, F (Phenylalanine, 6 carbons in sidechain) is a larger residue than L (Leucine, 4 carbons in sidechain), suggesting that the feature that causes 'discomfort' at this position is size; the bulkier residue unbalances the machine and favors the 'hold the Pi' state.

Given this simplistic model for what's going on, a straightforward prediction would be: shrinking L176 should have the opposite effect of enlarging it. Thus if L176F is the opposite of G680V, then L176A (alanine, 1 carbon sidechain) should be the opposite of the opposite of G680V, or the same as G680V. How can we test this genetically? Simple. L176F is not the only suppressor of G680V. Another is V192F. This mutant is defective on its own and requires (is suppressed by) G680V. If our logic above is correct, then L176A should be able to substitute for G680V and provide function in a molecule containing the V192F defect. This is indeed the case! All of this contributes to the following image that summarizes the genetic logic and relationships shown at left. This symbolism will be used throughout this section: the grey line indicates suppression; the double-headed arrow indicates that each gray circled mutant requires a mutant from the other gray circle for myosin function. The triangle indicates a residue where changes in size 'toggle' the motor to different states. Change to a smaller residue (small end of the triangle; orange) suppresses mutants in the indicated gray circle (T189I, V192F) while change to a larger residue (large end of the triangle; green) suppresses G680V.

We have a number of such findings in the works; they'll be appearing in this section 'soon'. Positions under investigation for this type of property include L529, V534 and A574. A similar class is found in the region 500-600, wherein MOST positive residues that change to negative suppress L596S, whereas MOST negative residues in this region, when changed to positive ones, suppress ÆWrinkle (itself a suppressor and suppressee of L596S).

The current Picture of Everything can be found HERE.

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Bruce Patterson http://research.biology.arizona.edu/myosin