The role of ATP, ADP and divalent cations in the formation of binary and ternary complexes of actin, cofilin and DNase I

Abstract

Actin is the major cytoskeletal protein of virtually all eukaryotic cells. Actin assembly/disassembly is involved in a variety of cellular processes and actin-binding proteins are essential in regulation of the pool of actin monomers. Cofilin and DNase I are actin-binding proteins, which form both binary (actin-DNase 1, cofilin-actin) and ternary (cofilin-actin-DNase I) complexes with actin. Here we use native gel electrophoresis to examine the roles of ATP, ADP, Ca2+ and Mg2+ in the formation of these complexes as well as on the ability of actin to self-assemble. Conditions which favour actin polymerisation are: ATP (no Me2+) > or = ADP (no Me2+) > ADP-Ca2+ = ADP-Mg2+ > ATP-Mg2+ > ATP-Ca2+. Preferential conditions for the formation of the binary actin-cofilin complex are: ADP-Mg2+ > or = ADP-Ca2+ >> ATP-Ca2+ approximately equals ATP-Mg2+ approximately equals ADP-No Me2+ approximately equals ATP-No Me2+. Actin forms a very tight complex with DNase I in the order: ATP-Ca2+ > or = ATP-Mg2+ approximately equals ADP-Mg2+ approximately equals ADP-Ca2+ > or = ADP-(no Me2+) > ATP-(no Me2+). Effectively, the complex does not form in the presence of ATP and the absence of free Me2+. Finally, the conditions which favour the formation of a ternary complex of cofilin-actin-DNase I resemble the actin-DNase I, namely: ATP-Ca2+ approximately equals ADP-Ca2+ approximately equals ADP-Mg2+ approximately equals ATPMg2+ ADP (no Me2+) > ATP-(no Me2+).

Mapping the ADF/Cofilin Binding Site on Monomeric Actin by Competitive Cross-linking and Peptide Array: Evidence for a Second Binding Site on Monomeric Actin

“…29 The existence of ternary complex demonstrates that the main interaction site of ADF or cofilin is the G-site. Binding does not occur at the F-site because DNase I binds monomeric actin very tightly and cannot be displaced by ADF or cofilin.…”

The distribution of cofilin and DNase I in vivo

“…Formation of a cofilin-actinDNase I ternary complex has been demonstrated in vitro using both native (non-denaturing) PAGE gels [17] and Phast gels [18]. These reports determined that cofilin and DNase I bind cooperatively to actin forming a stable complex that is fairly insensitive to buffer conditions.…”

“…Furthermore, we have demonstrated that the binding between cofilin, actin and DNase I in the ternary complex in vitro is essentially (Ca 2+ or Mg 2+ ) [17].Conversely, the cofilin-actin and actin-DNase I binary complexes are strongly influenced by buffer nucleotides and cations. The formation of the cofilin-actin-DNase I ternary complex has since been shown to be a cooperative process [18].…”

Structure and Function of Bovine Pancreatic Deoxyribonuclease I

“…Dos Remedios and colleagues found the binary or ternary complexes among DNase I, actin and cofilin which suggested that DNase I could act as a stabilizer of actin monomers by effectively removing them from the pool of monomers available for the filament assembly. Thus DNase I could be a natural modulator on the action of cofilin in cytoskeleton [39,40].…”