Intermolecular dynamics and function in actin filaments

Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments.     

Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments.

The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin.     

In vitro motility of skeletal muscle myosin and its proteolytic fragments

We have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KCl concentrations below a critical value (60 mM with myosin and HMM, 100 mM with S-1), and the sliding velocities were quite similar below the critical KCl concentration. However, at KCl concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were approximately 300 microM for S-1 and approximately 200 microM with HMM below the critical KCl concentration, and approximately 5,000 microM above the critical KCl concentration. This increase in KATPase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KCl concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.     

Disulfide cross-linking of caldesmon to actin.

Treatment of a solution of actin and smooth muscle caldesmon with 5,5'-dithiobis(2-nitrobenzoic acid) results in the formation of a disulfide cross-link between the C-terminal penultimate residue Cys-374 of actin and Cys-580 in caldesmon's C-terminal actin-binding region. Therefore, these 2 residues are close in the actin-caldesmon complex. Since myosin also binds to actin in the vicinity of Cys-374 and since caldesmon inhibits actomyosin ATPase activity by the reduction of myosin binding to actin, then the inhibition might be by caldesmon sterically hindering or blocking myosin's interaction with actin. [Ca2+]Calmodulin, which reverses the inhibition of the ATPase activity, decreases the yield of the cross-linked species, suggesting a weakening of the caldesmon-actin interaction in the cross-linked region. It is possible to maximally cross-link one caldesmon molecule/every three actin monomers, in the absence or presence of tropomyosin, clearly ruling out an elongated, end-to-end alignment of caldesmon on the actin filament in vitro, and raising the possibility that the N-terminal part of caldesmon projects out from the filament. Reaction of 5,5'-dithiobis(2-nitrobenzoic acid)-modified actin with caldesmon leads to the same disulfide cross-linked product between actin and caldesmon Cys-580, enabling the specific labeling of the other caldesmon cysteine, residue 153, in the N-terminal part of caldesmon with a spectroscopic probe.     

Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

Muscle actin cleaved by proteinase K: its polymerization and in vitro motility.

Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F-actin: Vmax = 0.24 s-1, and Kapp = 2.8 microM, while Vmax = 7.6 s-1, and Kapp = 13 microM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 microns/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.     

Removing the two C-terminal residues of actin affects the filament structure.

We define conditions under which the two C-terminal residues of actin, Cys-374 and Phe-375, can be selectively removed by proteolysis with trypsin. This modification had little effect on the secondary structure of actin detected by Fourier-transform infrared spectroscopy. However, removing these residues caused small but significant decreases in the critical concentration of actin, in its ability to activate myosin ATPase, and in its interaction with tropomyosin and troponin. Removing residues 374-375 caused dramatic changes in the actin filament as seen by electron microscopy. The filaments had a much greater and more irregular curvature and were intertwined into disordered multifilament bundles. Removing 374-375 also significantly lowered the flow viscosity of filamentous-actin solutions. These data suggest an increase in the flexibility and fragility of the filament, supporting the idea that the C-terminus forms one of the major intermonomer contacts in the filament.     

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.     

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Ga?azkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.     

The tert-butyl hydroperoxide-induced oxidation of actin Cys-374 is coupled with structural changes in distant regions of the protein.

The susceptibility of monomeric actin to both methionine and cysteine oxidation when treated with the oxidizing agent tert-butyl hydroperoxide (t-BH) was investigated. The results show that no methionine residue was susceptible to oxidation by t-BH at concentrations of 1-20 mM, while Cys-374, one of the five cysteine residues of the actin molecule, was found to be the site of the oxidative modification. Perturbations in the intrinsic tryptophan fluorescence and the decreased susceptibility to limited proteolysis by alpha-chymotrypsin and subtilisin of oxidized actin give an indication of some alterations in protein conformation in subdomain 1, and in the central segment of surface loop 39-51, in subdomain 2. Urea denaturation curves indicate a lower conformational stability for the oxidized actin. G-actin structural alterations due to Cys-374 oxidation produced by t-BH result in a decrease in the maximum rate of polymerization, an increase in both the delay time and the time required for half-maximum assembly, a decrease in the elongation rate, and enhancement of the critical monomer concentration for polymerization. The results suggest that oxidation of actin Cys-374 induces structural alterations in the conformation of at least two different distant regions of the molecule. The involvement of both the C-terminus of the actin polypeptide chain and the DNase-I-binding loop in the intermonomer interactions in the polymer could account for the altered kinetics of polymerization shown by the oxidized actin.     

Potentiated state of the tropomyosin actin filament and nucleotide-containing myosin subfragment 1

The main purpose of this study was to determine whether potentiation of acto-S-1 ATPase activity (activity higher than that obtained with tropomyosin-free actin) could be caused by nucleotide-containing acto-S-1 complexes. In addition, we wanted to know whether these complexes also have a positive cooperative effect on their own apparent binding constant under conditions where nucleotide-free acto-S-1 complexes cause potentiation of ATPase activity. Using calcium-saturated troponin-tropomyosin actin filaments, we observed potentiation of ATPase activity in the presence of 5.0 mM magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP) and calculated that the ability of acto-S-1-AMPPNP complexes to cause potentiation must have been very similar to that of nucleotide-free acto-S-1 complexes. In extension of earlier studies, potentiated acto-S-1 ATPase activity was characterized by an increase in Vmax and, as observed before, a lowering of the apparent Km for subfragment 1 (S-1). Under conditions similar to those that produce the potentiation of acto-S-1 ATPase activity, the apparent actin binding constant of nucleotide-free S-1 was increased about 3-5 fold while the apparent binding constant of AMPPNP to actin-bound S-1 was reduced to (2.5-10) x 10(2) M-1 compared to that of about (1-5) x 10(3) M-1 for S-1 bound to tropomyosin-free actin. Under the same conditions, the apparent binding constant of S-1-AMPPNP to actin was not increased. We suggest that a potentiated state of the tropomyosin actin filament is produced by the cooperative action of acto-S-1 or acto-S-1-AMPPNP complexes. The potentiated state is characterized by an increase in the Vmax of the acto-S-1 ATPase activity, increased binding constants for S-1 and S-1-ADP, and increased binding of tropomyosin to actin.     

Actin cross-linking and inhibition of the actomyosin motor.

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.     

Role of actin C-terminus in regulation of striated muscle thin filament

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca²⁺ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca²⁺, but it reduces tropomyosin-troponin affinity in the absence of Ca²⁺. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca²⁺-dependent regulation of the ATPase activity is preserved. Without Ca²⁺ the ATPase activity is fully inhibited, but in the presence of Ca²⁺ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.     

Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.     

Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces.

The topography of the rigor complex between subfragment-1 (S-1) of myosin and actin was investigated by using several specific antibodies directed to well-located sequences in actin. A major contact area for S-1 was characterized in the hydrophilic 18-28 constant sequence, and the variable 1-7 sequence was only found to be in close proximity to the interface. The C-terminal extremity of actin situated around Cys-374 appeared to be included in a region close to the S-1 heavy chain and the N-terminal part of actin. The interaction between tropomyosin and actin was also studied. Neither of the terminal parts of actin were involved in this interaction. Thus, the regions involved in the interactions of S-1 and tropomyosin with actin do not overlap.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Cofilin and DNase I affect the conformation of the small domain of actin

Cofilin binding induces an allosteric conformational change in subdomain 2 of actin, reducing the distance between probes attached to Gln-41 (subdomain 2) and Cys-374 (subdomain 1) from 34.4 to 31.4 A (pH 6.8) as demonstrated by fluorescence energy transfer spectroscopy. This effect was slightly less pronounced at pH 8.0. In contrast, binding of DNase I increased this distance (35.5 A), a change that was not pH-sensitive. Although DNase I-induced changes in the distance along the small domain of actin were modest, a significantly larger change (38.2 A) was observed when the ternary complex of cofilin-actin-DNase I was formed. Saturation binding of cofilin prevents pyrene fluorescence enhancement normally associated with actin polymerization. Changes in the emission and excitation spectra of pyrene-F actin in the presence of cofilin indicate that subdomain 1 (near Cys-374) assumes a G-like conformation. Thus, the enhancement of pyrene fluorescence does not correspond to the extent of actin polymerization in the presence of cofilin. The structural changes in G and F actin induced by these actin-binding proteins may be important for understanding the mechanism regulating the G-actin pool in cells.     

Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament.

We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate and (b) copolymerization of unmodified actin monomers with monomers cross-linked internally with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Each of these modifications abolished the interaction of the modified monomers with myosin, so the remaining interactions were exclusively with unmodified monomers. The two modifications had similar effects on the interaction of actin with myosin in solution: decreased affinity of myosin heads for unmodified actin monomers, without a change in the Vmax of actin-activated myosin ATPase activity. However, modification (b) produced much greater inhibition of actin sliding on a myosin-coated surface, as measured by an in vitro motility assay. These results provide insight into the functional consequences of cooperative interactions within the actin filament.     

Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1

Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.