title: Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.
abstract: Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an alpha-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rould
authors.forename: Qun
authors.initials: Q
authors.lastname: Wan
authors.forename: Peteranne B
authors.initials: PB
authors.lastname: Joel
authors.forename: Susan
authors.initials: S
authors.lastname: Lowey
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Trybus
mesh_terms: D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004330:Drosophila; D006868:Hydrolysis; D007313:Insecta; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020860:Subtilisin / Q000737:chemistry
chemical_list: D000199:Actins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D020860:Subtilisin