Carl Miller

Structural connectivity in actin: effect of C-terminal modifications on the properties of actin

In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.

Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262–274 ACTIN-ACTIN AND ACTIN-MYOSIN INTERACTIONS IN ACTIN FILAMENTS

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 Å 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.

Mutational Analysis of the Role of the N Terminus of Actin in Actomyosin Interactions. Comparison with Other Mutant Actins and Implications for the Cross-Bridge Cycle†

Yeast actin mutants with acidic residues at the N terminus either neutralized (DNEQ) or deleted (delta-DSE) were used to assess the role of N-terminal acidic residues in the interactions of actin with myosin in the contractile cycle. Cosedimentation experiments revealed an approximately 3-fold decrease in the binding constant for DNEQ and delta-DSE actins to myosin subfragment-1 (S1) relative to that of wild type actin both in the presence of MgATP and in the absence of nucleotides (strong binding). DNEQ and delta-DSE actins protected S1 from tryptic digestion as well as the wild type and rabbit actins. The activation of S1 ATPase by DNEQ and delta-DSE actins (up to 50 microM) was very low but increased greatly after cross-linking these mutant actins to S1 by dimethyl suberimidate. Thus, the increased dissociation of mutant actins from S1 in the presence of ATP is the main cause for the low acto-S1 ATPase activities. At low-ionic strength conditions and in the presence of methylcellulose, the DNEQ and delta-DSE actins moved in the in vitro motility assays at a mean velocity similar to that of wild type actin (3.0 microns/s). Yet, the sliding velocity of the N-terminal and D24A/D25A and E99A/E100A mutant actins decreased relative to that of the wild type at all levels of external load introduced into the assay and at low densities of heavy meromyosin (HMM) on the cover slip. This indicates a lower relative force generation with the mutant actins. In contrast, the force generated under the same conditions with the 4Ac mutant actin (with four acidic charges at the N terminus) was higher than with wild type actin. At higher-ionic strength conditions (I = 150 mM), the sliding of the DNEQ and delta-DSE as well as that of the D24A/D25A and E99A/E100A actins ceased even in the presence of methylcellulose, while I341A actin (deficient in strong binding to myosin) still moved. These results indicate the importance of electrostatic actomyosin interactions under physiological salt conditions and show functionally distinct roles for the different myosin binding sites on actin.

Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

A novel kDa form of subtilisin cleaved actin: structural and functional consequences of cleavage between Ser234 and Ser235

A new 27/16 kDa form of cleaved actin was prepared by subtilisin cleavage between Ser234 and Ser235 of F(MgADP)‐actin complexed with BeFx. The cleavage had little effect on actin‐actin interactions as probed in polymerization measurements and by electron microscopy. In circular dichroism melting experiments the thermostability of F‐actin was reduced by about 10°C by this cleavage. The in vitro motility and V max, but not K m, of actomyosin ATPase were decreased by about 20% upon 27/16 kDa cleavage of F‐actin. The binding of tropomyosin to actin was unchanged by this modification.