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Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.

Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity, the actin cross-linking domain (ACD), has recently been identified. In this study, we further investigate the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independent of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of Lethal Factor from B. anthracis, LFNACD, has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determine that LFNACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg2+ and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.

According to the Lorenz et al. (Lorenz, M., Poole, K. J., Popp, D., Rosenbaum, G., and Holmes, K. C. (1995) J. Mol. Biol. 246, 108–119) atomic model of the actin-tropomyosin complex, actin residue Asp-311 (Glu-311 in yeast) is predicted to have a high binding energy contribution to actin-tropomyosin binding. Using the yeast actin mutant E311A/R312A in the in vitro motility assays, we have investigated the role of these residues in such interactions. Wild type (wt) yeast actin, like skeletal α-actin, is fully regulated when complexed with tropomyosin (Tm) and troponin (Tn). Structure-function comparisons of the wt and E311A/R312A actins show no significant differences between them, and the unregulated F-actins slide at similar speeds in thein vitro motility assay. However, in the presence of Tm and Tn, the mutation increases both the sliding speed and the number of moving filaments at high pCa values, shifting the speed-pCa curve nearly 0.5 pCa units to the left. Tm alone (no Tn) inhibits the motilities of both actins at low heavy meromyosin densities but potentiates only the motility of the mutant actin at high heavy meromyosin densities. Actin-Tm binding measurements indicate no significant difference between wt and E311A/R312A actin in Tm binding. These results implicate allosteric effects in the regulation of actomyosin function by tropomyosin.

Residues 262–274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys374 or Cys265. These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was ∼50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 A of one another. Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.

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.

The rates of tryptic digestion of the 50/20-kDa junction in myosin in cardiac myofibrils were determined under various solvent conditions. This cleavage reaction is slow in the rigor solvent and proceeds at a fast rate in the presence of MgATP. When the reaction solvent contains 50% ethylene glycol, the digestion of myosin in the presence of MgATP occurs at the same rate as in myofibrils relaxed by Mg adenyl-5'-yl imidodiphosphate (AMP-PNP). It is shown that with the help of two reference rates of digestion, for attached and dissociated myosin heads, the initial cleavage rates of myosin in the presence of nucleotides accurately measure the dissociation of cross-bridges from actin in myofibrils. Under physiological salt conditions and at 24 degrees C, MgADP, MgPPi, and MgAMP-PNP cause only small cross-bridge detachment (less than or equal to 15%) in cardiac myofibrils. The dissociation of myosin from actin is greatly increased by lowering the solvent temperature to 4 degrees C. Lowering the salt concentration of the solvent from 0.1 to 0.01 M NaCl has the most pronounced effect on the rates of myosin digestion in the presence of MgATP. In the low salt medium a substantial fraction of myosin heads (at least 30%) appears to be attached to actin in the presence of 5 mM MgATP.

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin subfragment 1 (S-1) were determined for free S-1, acto-S-1, and acto-S-1 in the presence of magnesium adenyl-5'-yl imidodiphosphate (Mg AMP-PNP) and MgATP under ionic strength conditions ranging from 30 to 124 mM. The percentage of S-1 bound to actin in the presence of Mg AMP-PNP and MgATP was calculated from these rates for each set of digestion experiments. Parallel experiments carried out in an Airfuge centrifuge on identical acto-S-1 solutions yielded independent information on the binding of S-1 to actin. The results of binding measurements by these two methods were in excellent agreement in all cases tested, covering the range from 15 to 95% binding of S-1 to actin. Tryptic digestions of synthetic mixtures of S-1 and p-phenylenedimaleimide S-1 in the presence of actin demonstrated that a two-component system of myosin heads with different affinities for actin can be resolved into its constituents by the proteolytic rates method. The results of this work justify applications of the proteolytic rates method to actomyosin binding studies in more complex systems.

The ionic strength dependence of the absolute activation of myosin MgATPase by actin binding or by chemical modification of the SH1 sulfhydryl group has been investigated. At physiological ionic strengths the extent of activation induced by the two procedures are essentially identical, but differ markedly as the ionic strength is decreased below 0.10. Similar behavior is also found for subfragment I. These findings are discussed in terms of the mechanism of actin activation of myosin MgATPase under physiological conditions.