Allosteric Communication Pathway

In dynein we have made an intriguing observation: while the ATPases are located at modules in AAA+ ring, the MTBD domain, which binds protein to microtubule using energy from ATP hydrolysis, is situated at opposite end of stalk. This makes one wonder about mechanism of energy flow over such long distance.

In a normal protein, the domain providing energy is generally in close proximity with the domain consuming energy for optimal efficiency in energy flow. However, such is not the case for dynein. Hence, it is apparent that some structures between the aforementioned parties may involve in bilateral allosteric communication.

b-factor - to reflect molecule flexibility

Comparisons of stalk-strut structures in two peptides of WT and ΔMTBD found its high structural flexibility in presence and absence of MTBD, which may indicate its role in communication relaying. Upon closer inspection, there is an extensive network of interactions between stalk and strut, further suggesting strut’s involvement in the pathway. To confirm this, a strut mutant (MD) was generated having replaced by long glycine chain, eliminating all existing interactions with stalk. Kinetic analysis results in ATPase activity equivalent to wildtype kcat (~90 s-1) but reduced microtubule binding affinity (Kd > 10μM).

The logic is that ATP hydrolysis maintains suitable microtubule binding affinity. In the case of MD, ATP continues to hydrolysis and give out energy as usual, but it no longer has effect on the binding affinity. So, something has gone wrong, and that thing is the strut. (waste of energy lol)

The above shows that the pathway is disrupted by mutation of strut, reflecting significance of stalk-strut interactions in proper communication between ATPase and MTBD.

Next, we want to know what domains involved in pathway between the stalk and AAA1 ATPase. Among all modifications of different domains between the two, deletion of segment of C-sequence leads to both reduced ATPase activity and microtubule binding affinity. This shows no only C-sequence is involved in allosteric communication pathway, it influences proper function of ATPase. Knowing that part of the sequence is inessential in motor activity, H1 helix would be the one for communication by elimination.

H1 helix of C-sequence serves as bridge linking AAA1-AAA6 segment to H6 helix of AAA5 where strut extends and reaches stalk, in between of AAA5 alpha submodule and AAA5 extension

Summary of Allosteric Communication