Non-polymerizable monomeric actin: Difference between revisions

Non-polymerizable monomeric actin or AP-actin is an Sf9-expressed cytoplasmic actin harboring two point mutations that prevent the monomer from polymerizing into actin filaments. These mutations allow for the crystallization of actin without the use of specific toxins or actin-binding proteins that may influence the structure. The crystal structure of AP-actin has been solved for the ADP-bound form ([[2HF3]]) and the ATP-bound form ([[2HF4]])<ref>PMID: 16920713</ref>. These two structures are shown below as a morph between the two states.

There is some controversy over whether or not the D-loop undergoes structural changes upon actin binding ATP. In the structure of AP-actin, the D-loop is disordered in both the ATP and ADP-bound state. Also, there is no evidence that structural changes in the nucleotide binding cleft propagate to subdomain 2. This argues that the D-loop remains disordered in both states. However, other groups show large ATP-dependent structural changes in the D-loop<ref>PMID:11474115</ref>. This is illustrated, right, in a subdomain 2 morph of actin complexed with tetramethylrhodamine (TMR) in the ADP-bound state, [[1J6Z]] and actin complexed with DNAase I in the ATP-bound state, [[1ATN]]. These structures revile that the D-loop is disordered when actin is bound to ATP, and transitions to an alpha-helix in the ADP-bound state. It has been suggested that the alpha helix in the ADP-bound state results from crystal packing. In support of this, actin complexed with TMR in the ADP state shows extensive neighboring contacts around the D-loop (shown left, lower panel). In contrast, AP-actin, [[2HF3]], shows far fewer crystal contacts around the D-loop (shown left, upper panel). These crystal contacts have been proposed to result in the nucleotide-dependent structural changes in the D-loop observed in some structures. In further support of this, the sequence of the D-loop, HQGVMVGMG, has a low propensity to form an alpha helix. However, molecular dynamic simulations show that the D-loop favors the alpha helix conformation in the ADP state, and not the ATP or ADP-Pi states. This study supports a model where small perturbations in the active site shift the equilibrium of the D-loop between the coil and helix state. Further studies are needed resolve these conflicting reports.