Non-polymerizable monomeric actin

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). These two structures are shown below as a morph between the two states.

Structural features of actinStructural features of actin

The actin monomer, or , contains four structural domains: (residues 1-32, 70-144 and 138-375), (residues 33-69), (residues 145-180 and 270-337) and (residues 181-269). These domains can be classified as largely alpha/beta connected by loops which are shown in some structural analysis to undergo significant nucleotide dependent structural changes. The is located in domain 2 (residues 40-51). This loop is not shown in the crystal structure of AP-actin because it was found to be disordered in both the ATP and ADP-bound state, conflicting with other reports. This is discussed in more detail below. The or sensor loop is located in domain 1 (residues 70-78) and is though to contain important residues for sensing the nucleotide state. The (residues 165-172) is located in domain 3 and is important for the binding of WH2 domain-containing proteins. The (residues 11-16) and the (residues 154-161) are contained in the nucleotide-binding cleft.

AP-actin is a cytoplasmic actin encoded from the Drosophila melanogaster 5C actin gene. It shares 98.7% sequence homology with human γ-cytoplasmic actin. AP-actin contains two point mutations, that render it incapable of polymerizing into actin filaments. These mutations were shown to have little effect on ATP hydrolysis or long range structural changes.

Nucleotide-dependent structural changes in the nucleotide-binding cleft of AP-actinNucleotide-dependent structural changes in the nucleotide-binding cleft of AP-actin

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	Morph of AP-actin showing conformational changes between actin bound to ATP (2HF4) and ADP (2HF3). Nucleotide is not shown.

File:Sensor loop png.png

Sensor loop of AP-actin bound to ADP (grey) and ATP (white)

Structural changes between the ATP and ADP-bound state of are confined to the active site. The of the two states outside the sensor loop region superimpose very well, with a RMSD of 0.19 angstroms. A close-up of the reviles how binding ATP elicits structural changes in the nucleotide-binding cleft that is propagated to the sensor loop. When actin is bound to ADP, the serine-14 side chain forms a hydrogen bind with the beta phosphate ADP. Upon ATP binding, the gamma phosphate sterically clashes with the oxygen of serine-14, forcing the hydroxyl to rotate 130°. The displaced serine residue impinges on the backbone carbonyls of residues isoleucine-71 and glutamine-72 in the sensor loop. This causes a 180° rotation of the peptide linkage between glutamine-72 and histidine-73. These structural changes impact residues proximal to the sensor loop. Transition to the ATP-bound state causes glutamine-72 to form a new hydrogen bond with threonine-77. This induces a reorientation of . ATP binding also disrupts hydrogen bonding between arginine-183 and residues 72 and 73 of the sensor loop causing a conformational change of arginine-183. Aspartic acid-179 stacks against histidine-73 in the ATP-bound state which induces a shift in the position of arginine-177 to improve salt bridge formation with aspartic acid-179.

About this StructureAbout this Structure

This is where I will add my text. 2hf4 is a 1 chain structure of Actin with sequence from Drosophila melanogaster. Full crystallographic information is available from OCA.

See AlsoSee Also

• Actin