Non-polymerizable monomeric actin: Difference between revisions

{{STRUCTURE_2hf4| PDB=2hf4 | SCENE=User:Thomas_E_Sladewski/Sandbox_1/2hf4_blue_helix2/1|CAPTION= Crystal structure of the AP-actin in complex with ATP,  [[2HF4]]. Alpha helices are in green. Loops and beta strands are in blue. ATP is designated with gold spheres and Ca+2 ions in red spheres.  }}  

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.

<table width='450' align='left' cellpadding='5'><tr><td rowspan='2'>&nbsp;</td><td bgcolor='#d0d0d0'><applet load=Image:2HF3 2HF4 10state morph4.pdb' size='450' align='right' scene='User:Thomas_E_Sladewski/Sandbox_1/10state_morph_scene2/2' /></td></tr><tr><td bgcolor='#d0d0d0'>Morph of AP-actin showing conformational changes between actin bound to ATP ([[2HF4]]) and ADP ([[2HF3]]). Nucleotide is not shown.</td></tr></table>

[[Image:2hf4 d loop crystal packing.png|250px|left|thumb| Crystal packing interactions between ADP-bound AP-actin, 2HF3, (grey ribbon) and neighboring residues in subdomain 2 of an adjacent monomer in the crystal within 15 angstroms of the D-loop in the unit cell (blue ribbons). Residues 39-50 are shown in red.]]

[[Image:1J6Z d loop crystal packing.png|250px|left|thumb| Crystal packing interactions between ADP-actin, 1J6Z, complexed with TMR (grey ribbon) and neighboring residues in subdomain 2 of an adjacent monomer in the crystal within 15 angstroms of the D-loop in the unit cell (blue ribbons). Residues 39-50 are shown in red.]]

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 name="TMR">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]]<ref name="TMR"/>. 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.  

Residues 70-77 in subdomain 1 make up the H-loop. This loop is also known as the sensor loop because structural changes in the nucleotide binding cleft, such as tortional movement of serine 14, are propagated to these amino acids. Thus, the H-loop undergoes the largest nucleotide-dependent conformational changes as seen above. The H-loop is a good example of a type I’ β-turn, shown left. β-turns are comprised of four consecutive residues whose distance between the α-carbons of residue i and i+3 is less than 7 Å. Typically, β-turns form hydrogen bonds between the carboxyl group of residue i and the amide group of residue i+3. Here, the carbonyl of glutamic acid-72 (i) forms a hydrogen bond with the amide group of isoleucine 75 (i+3). In addition, another hydrogen bond is formed between the carbonyl of isoleucine 75 and the amide of glutamic acid 72, indicating a capping box motif. β-turns can be further classified by the dihedral angles of the i+1 and i+2 residues as well as the distance between the α-carbons of residue i and i+3. This turn is consistent with a type I’ β-turn.

Helix capping describes the interruption of hydrogen bonds between amide hydrogen’s and carbonyl oxygen’s at the termini of an α-helix. This occurs when amide hydrogen’s form hydrogen bonds with alternative binding partners such as side chains. A number of helix capping motifs have been described to form these alternative hydrogen bonding patterns. The N-terminus of α-helix eight (residues 202-217) in AP-actin is capped by a “capping box” motif. This motif caps two (threonine-202 and glutamic acid-205) of the initial four amide hydrogen donors in the helix, shown right. <ref name="Aurora">PMID: 9514257</ref>