The Interaction of Caldesmon with the COOH Terminus of Actin

Caldesmon interacts with the NH2-terminal region of actin. It is now shown in airfuge centrifugation experiments that modification of the penultimate cysteine residue of actin significantly weakens its binding to caldesmon both in the presence and absence of tropomyosin. Furthermore, as revealed by fluorescence measurements, caldesmon increases the exposure of the COOH-terminal region of actin to the solvent. This effect of caldesmon, like its inhibitory effect on actomyosin ATPase activity, is enhanced in the presence of tropomyosin. Proteolytic removal of the last three COOH-terminal residues of actin, containing the modified cysteine residue, restores the normal binding between caldesmon and actin. These results establish a correlation between the binding of caldesmon to actin and the conformation of the COOH-terminal region of actin and suggest an indirect rather than direct interaction between caldesmon and this part of actin. Originally published Journal of Biological Chemistry, Vol. 266, No. 30, Oct 199

Catastrophic disassembly of actin filaments via Mical-mediated oxidation.

Actin filament assembly and disassembly are vital for cell functions. MICAL Redox enzymes are important post-translational effectors of actin that stereo-specifically oxidize actin's M44 and M47 residues to induce cellular F-actin disassembly. Here we show that Mical-oxidized (Mox) actin can undergo extremely fast (84 subunits/s) disassembly, which depends on F-actin's nucleotide-bound state. Using near-atomic resolution cryoEM reconstruction and single filament TIRF microscopy we identify two dynamic and structural states of Mox-actin. Modeling actin's D-loop region based on our 3.9 Å cryoEM reconstruction suggests that oxidation by Mical reorients the side chain of M44 and induces a new intermolecular interaction of actin residue M47 (M47-O-T351). Site-directed mutagenesis reveals that this interaction promotes Mox-actin instability. Moreover, we find that Mical oxidation of actin allows for cofilin-mediated severing even in the presence of inorganic phosphate. Thus, in conjunction with cofilin, Mical oxidation of actin promotes F-actin disassembly independent of the nucleotide-bound state

Solution Properties of Tetramethylrhodamine-Modified G-Actin

In the recently solved structure of TMR-modified ADP-G-actin, the nucleotide cleft is in a closed state conformation, and the D-loop contains an α-helix (L. R. Otterbein, P. Graceffa, and R. Dominguez, 2001, Science, 293:708–711). Subsequently, questions were raised regarding the possible role of the TMR label on Cys(374) in determining these aspects of G-actin structure. We show here that the susceptibility of D-loop on G-actin to subtilisin cleavage, and ATP/ADP-dependent changes in this cleavage, are not affected by TMR-labeling of actin. The TMR modification inhibits nucleotide exchange, but has no effect on DNase I binding and the fast phase of tryptic digestion of actin. These results show an absence of allosteric effects of TMR on subdomain 2, while confirming ATP/ADP-dependent changes in D-loop structure. In conjunction with similar results obtained on actin-gelsolin segment 1 complex, this works reveals the limitations of solution methods in probing the putative open and closed nucleotide cleft states of G-actin

Drebrin inhibits cofilin-induced severing of F-actin.

Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments

Drebrin inhibits cofilin-induced severing of F-actin.

Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments