This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Actin is a highly conserved eukaryotic protein which polymerizes into filaments that form a major component of the cytoskeleton. The actin fold consists of four subdomains (a total MW of about 40kD) and is shared by a number of other actin-related proteins (Arps), which have homolgous sequences and presumably the same fold as actin. Actin and the most actin-like Arps bind and hydrolyze ATP. In the case of actin, the dissociation of the gamma phosphate is thought to produce a conformational change which creates critical differences in the biochemical properties of actin, depending on which adenine nucleotide is bound. ADP-actin and ATP-actin monomers, for instance, show markedly different polymerization properties, and ADP- and ATP-actin filaments have different affinities for certain filament binding proteins; these differences are exploited by the cell in order to control actin dynamics. Arp3 is an actin-related protein which is part of the Arp2/3 complex, a protein assembly that nucleates actin filaments in response to extracellular signals. In vito FRET experiments have shown that ATP-binding causes a conformational change in Arp3 that is necessary for the function of the complex. Crystal structures of actin with either ADP or ATP bound have shown that subtle changes occur in the nucleotide binding cleft after phosphate dissociation, and that these changes are relayed to the DNAse binding loop, which undergoes a unfolded to alpha helix transition. In contrast, electron microscopy showed that in the context of the filament, actin adopts an open conformation when ADP is bound and a closed conformation when ATP is bound. Recent crystal structures of Arp2/3 complex supported this idea; ATP binding to Arp3 caused the cleft to close, while ADP bound to an open cleft. We seek to understand the relationship between nucleotide-binding and conformation using the available crystal structures of ATP-actin (closed), ADP-actin (closed), apo-Arp3 (open), ADP-arp3 (open), and ATP-arp3 in molecular dynamics (MD) simulations. In our first set of simulations we will keep the nucleotide-binding state the same as in the crystal structure and look for conformational changes. Our second round of simulations will involve changing the nucleotide binding state in a given structure and running a 5ns simulation to determine how changing the nucleotide affects conformation. We will look for rigid body motions that result in cleft opening or closing and for more local changes in the cleft and compare these to the available crystallographic snapshots. By clarifying the relationship between nucleotide-binding and conformation, we hope to understand both the mechanism of polymerization and depolymerization of actin and nucleation of actin filaments by Arp2/3 complex.
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