Conformational Changes In Actin Research Articles

A conformational change in F-actin when myosin binds: fluorescence resonance energy transfer detects an increase in the radial coordinate of Cys-374.

Interactions of myosin with actin filaments probably induce conformational changes in actin which are crucial for its function. Fluorescence resonance energy transfer spectroscopy can determine changes in distance (range 10-100 A) between two probes and therefore can sense conformational changes in proteins. We have investigated changes in the radial coordinates of fluorescent probes bound to Cys-374 of F-actin when either of the isozymes (S1A1 and S1A2) of myosin subfragment 1 (S-1) bind. Using 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide as donor and acceptor probes, respectively, we calculated a radius of 13-14 A. This distance increased by approximately 4.5 A upon addition of S-1. No differences were detected between the effects of S1A1 and S1A2. This increase is reversed by MgATP. The average position of the probes on Cys-374 is closer to the filament axis than expected from the current models of F-actin. S-1 increases the radial position of Cys-374 either by direct interaction or via an allosteric conformational change associated with its binding.

Cooperativity in F-Actin: Binding of Gelsolin at the Barbed End Affects Structure and Dynamics of the Whole Filament

We have studied the effect of gelsolin, a Ca-dependent actin-binding protein, on the microsecond rotational dynamics of actin filaments, using time-resolved phosphorescence (TPA) and absorption anisotropy (TAA) of erythrosin iodoacetamide attached to Cys374 on actin. Polymerization of actin in the presence of gelsolin resulted in substantial increases in the rate and amplitude of anisotropy decay, indicating increased rotational motion. Analysis indicates that the effect of gelsolin cannot be explained by increased rates of overall (rigid-body) rotations of shortened filaments, but reflects changes in intra-filament structure and dynamics. We conclude that gelsolin induces (1) a 10° change in the orientation of the absorption dipole of the probe relative to the actin filament, indicating a conformational change in actin, and (2) a threefold decrease in torsional rigidity of the filament. This result, which is consistent with complementary electron microscopic observations on the same preparations, directly demonstrates long-range cooperativity in F-actin, where a conformational change induced by the binding of a single gelsolin molecule to the barbed end is propagated along inter-monomer bonds throughout the actin filament.

Comparison of the Effects of Calponin and a 38-kDa Caldesmon Fragment on Formation of the “Strong-Binding” State in Ghost Muscle Fibers

We studied the conformational changes in actin filaments induced by the binding of calponin or a 38-kDa fragment of caldesmon, two actin-binding proteins known to inhibit actin-activated ATP hydrolysis by phosphorylated smooth muscle myosin. The F-actin in myosin-free muscle fibers (ghost fibers) was labeled with fluorescein-5-maleimide and the conformational change in actin was determined by polarized fluorimetry. Data show that both calponin and the 38-kDa caldesmon fragment inhibit the conformational changes in F-actin that are compatible with the “strong-binding” state between myosin heads and actin. Tropomyosin slightly reduces the effect produced by calponin, but enhances the effect produced by the 38-kDa caldesmon fragment.

Opposite Effects of Myosin Subfragment 1 on Binding of Cardiac Troponin and Tropomyosin to the Thin Filament

To better understand the regulation of striated muscle contraction, the effects of myosin subfragment 1 (S-1) on the actin binding of cardiac troponin and tropomyosin were investigated. Troponin's affinity for actin-tropomyosin was 4-fold stronger in the absence than in the presence of myosin S-1. CaCl2 had no effect on troponin binding to the thin filament in the presence of myosin S-1. The binding curve was weakly cooperative, implying interactions between adjacent troponin molecules. Myosin S-1 increased (40-200-fold) the affinity of tropomyosin for the thin filament, an effect opposite to the effect of myosin on troponin. This effect was highly cooperative and occurred in the presence of ADP or in the absence of nucleotide. Myosin altered the effect of ionic conditions on tropomyosin-actin binding, consistent with tropomyosin binding to a different site on F-actin in the presence of myosin. The results indicate that troponin-tropomyosin and strongly binding myosin cross-bridges do not compete for an F-actin binding site. Although repositioning of troponin-tropomyosin on the actin filament may be sterically required for tight myosin-actin binding, a myosin-induced conformational change in actin provides a better explanation for the complex effects of myosin on thin filament assembly.

Modulation of actin conformation and inhibition of actin filament velocity by calponin.

Calponin, an actin/calmodulin-binding protein present in smooth muscle thin filaments, modulates the actin-myosin interaction and actomyosin ATPase activity of smooth muscle myosin II. Binding of myosin heads to actin under conditions that produce weak or strong binding induces conformational changes in actin. Polarized fluorimetric measurements of rhodamine-phalloidin complex and 1,5-IAEDANS specifically linked to actin in myosin-free muscle fibers (ghost fibers) and to Cys-707 in myosin head, respectively, revealed conformational changes, as determined from the changes in orientation and mobility of fluorescent probes, upon addition of calponin to ghost fibers. The effect of calponin on conformational changes produced upon binding of phosphorylated or dephosphorylated heavy meromyosin (HMM) was also determined. Subfragment-1 preparation modified with NEM (NEM-S1) or pPDM (pPDM-S1) were used as models of strong and weak binding, respectively. Calponin changed both the orientation of fluorophores on the actin and the flexibility of the actin filaments, as determined from the angle between an actin filament and the fiber axis. Changes in the flexibility of actin filaments and the orientation of fluorophores produced by phosphorylated smooth muscle HMM were similar to those seen with NEM-S1, which formed a strong-binding association with actin and caused the transition of actin monomers to the "on" state; calponin markedly inhibited this effect. In contrast, pPDM-S1 and dephosphorylated HMM induced weak binding and the transition of actin monomers to the "of" state, and these effects were enhanced by calponin. Furthermore, calponin decreased the velocity of actin filament movement over skeletal muscle myosin O gamma phosphorylated smooth muscle myosin heads in an in vitro motility assay. These results suggest that calponin induces modulation of smooth muscle contraction by inhibiting the force-producing (strong-binding) state of cross-bridges and involves changes in actin conformation.

Actin interaction with purified dystrophin from electric organ of Torpedo marmorata: possible resemblance with filamin-actin interface.

We have purified dystrophin from Torpedo marmorata electric tissue by means of alkaline extraction in conjunction with an affinity chromatography column using anti-peptide antibodies. Using solution (cosedimentation) and solid phase experiments (sedimentation with Sepharose filamentous actin and ELISA), we have demonstrated that purified dystrophin is able to bind filamentous and monomeric actin. Using ELISA coupled with biotin labelled peptides and taking advantage of strong affinity binding of streptavidin-biotin complex, we have identified two exposed sequences of the actin molecule implicated in dystrophin binding: fragment 40-113, further restricted to peptide 75-106 and peptide 360-372. In a previous study, we have shown that fragment 40-113 displays binding site(s) for filamin but probably not for alpha-actinin. Moreover, we have recently reported that alpha-actinin and filamin display divergent behaviours towards conformational changes of actin. In this study, we have demonstrated that, similarly to filamin, dystrophin binding is insensitive to the locking of actin in a monomeric conformation. Taken together, these results lead us to favour the idea that dystrophin could share properties in common with filamin in its binding of actin.

Characterization of the actin binding site on smooth muscle filamin.

We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D. R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134) and the actin counterpart composed of residues 112-125 and 360-372 (we have already implicated) could constitute the main interface between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.

Differential binding of acapsulate and encapsulated strains ofCryptococcus neoformansto human neutrophils

Investigations into mechanisms of binding of encapsulated and acapsulate strains of Cryptococcus neoformans by human neutrophils were performed, using a monolayer assay. The two strains bound to neutrophils by different mechanisms although both had an absolute requirement for opsonization with complement components in normal human serum for binding to occur. Neutrophil binding of encapsulated yeasts required conformational changes in actin yet did not appear to lead to phagocytosis of the organism. A maximum of 12 acapsulate cells bound per neutrophil compared with only four encapsulated yeasts. Cytochalasin D treatment reduced the maximum numbers able to bind per neutrophil by 50%. The encapsulated cryptococci appeared to compete with each other for binding to neutrophils whereas the acapsulate yeast cells bound to neutrophils in an approximately Poisson distribution, suggesting independent binding. Binding of acapsulate cryptococci did not require actin filaments and appeared to trigger phagocytosis. Thus, the capsule of C. neoformans appeared to inhibit binding and internalization by neutrophils.

Conformational changes of actin induced by calponin

Calponin, an actin-linked regulatory protein in smooth muscle, caused a remarkable change in the fluorescence intensity of pyrene-labeled actin in the filamentous form. Calponin, an equimolar ratio to actin, decreased the fluorescence intensity of pyrene-labeled F-actin by some 60% to the level near monomeric actin. This change was partially reversed by Ca2+, when calmodulin was present. Thus it appears that calponin causes conformational changes in actin molecules in an actin filament so as to inhibit their interactions with myosin.

Detection of conformational changes in actin by fluorescence resonance energy transfer between tyrosine-69 and cysteine-374

The distance between 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride or DNS-Cl) attached to Tyr-69 and N-[[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) or N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys-374 in an actin monomer was measured to be 2.51 nm or 2.27 +/- 0.04 nm, respectively, by fluorescence resonance energy transfer. This distance does not change significantly when the actin monomer binds DNase I, when the monomer is polymerized, when the polymer interacts with myosin subfragment 1, or when it interacts with tropomyosin-troponin in the presence and absence of Ca2+. Changes in the distance were within 0.1 nm. The results indicate that the structure of the region involving Tyr-69 and Cys-374 is substantially rigid. A large blue shift (about 15 nm) of the fluorescence spectrum and a large increase (about 80%) in the fluorescence intensity of DNS-actin were observed when DNS-actin was denatured upon addition of EDTA. On the other hand, a red shift (about 7 nm) of the fluorescence spectrum and a large decrease (about 50%) in the fluorescence intensity were observed when DNS-actin was completely unfolded in 8 M urea. The results indicate that dansyl chromophore becomes less exposed to the aqueous environment by EDTA denaturation in contradiction to the case of intrinsic tryptophan residues in G-actin. Resonance energy transfer measurements showed that the distance between probes attached to Tyr-69 and Cys-374 on an actin monomer changes by 0.37 nm during EDTA denaturation, but that the distance becomes longer than 4.0 nm in 8 M urea in which no energy transfer is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin.

We studied the receptors on human cultured macrophages (MO-M phi) responsible for binding encapsulated and isogenic mutant acapsular strains of Cryptococcus neoformans, and whether such binding leads to a phagocytic event. Both strains required opsonization with complement components in normal human serum in order for binding to occur. Binding of the acapsular, but not the encapsulated, strain led to phagocytosis. MAb directed against any of the three defined complement receptors (CR) on MO-M phi (CR1, CR3, and CR4) profoundly inhibited binding of serum-opsonized encapsulated (and to a lesser extent acapsular) organisms to MO-M phi. Immunofluorescence studies demonstrated migration of CR to the area of the cryptococcal binding site. Trypsin and elastase inhibited binding of encapsulated and, to a lesser extent, acapsular yeasts to MO-M phi. Binding of encapsulated C. neoformans was profoundly inhibited by incubation in the cold or by inhibitors of receptor capping and actin microfilaments. Thus, multiple CR appear to contribute to binding of serum-opsonized encapsulated C. neoformans by MO-M phi. Binding is an energy-dependent process that requires conformational changes in actin yet does not lead to phagocytosis of the organism. In contrast, energy is not required for binding of acapsular yeasts by MO-M phi and binding triggers phagocytosis.

Propagation of Acto-S-1 ATPase Reaction-Coupled Conformational Change in Actin along the Filament1

F-Actin was partially cross-linked to myosin subfragment-1 (S-1) at various molar ratios (r = S-1/actin) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked acto-S-1 ATPase showed so called "super-activation," Vx. S-1 was added further to the cross-linked acto-S-1 and the ATPase activity, Vy, was measured. Since the added S-1 can interact only with the bare actin protomers within the cross-linked actin filament, the difference, delta V = Vy - Vx - Vs (where Vs is the ATPase activity of the additional S-1 alone), can indicate the state of the bare actin protomers while the cross-linked acto-S-1 is hydrolyzing ATP. With increasing r, delta V decreased much more rapidly than delta Vo(1 - r) (where delta Vo is delta V at r = 0) and reached a minimum around r = 0.15. As r increased further, delta V approached the level of delta Vo(1 - r). When SH1/SH2-blocked S-1 was cross-linked to F-actin, delta V decreased according to delta Vo(1 - r). Therefore, the large reduction of delta V, observed when intact S-1 was cross-linked, was coupled to the high ATPase activity of the cross-linked acto-S-1. Combining these data with other kinetic data, we could deduce that structural distortion in a cross-linked actin induced by the ATPase reaction of the S-1 partner propagated over several bare actin protomers along the filament and reduced their affinity for the S-1-ADP-Pi complex.(ABSTRACT TRUNCATED AT 250 WORDS)

PH-induced changes in G-actin conformation and metal affinity.

Metal-induced conformational changes in actin at 20 degrees C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+ concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+ concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+ binding to specific sites of moderate affinity [Zimmerle, C.T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545-6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+ addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+ concentrations (less than 200 microM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+ addition to Ca2+-actin are not associated with changes in the Mg2+ affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+ binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of phalloidin with chemically modified actin.

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60, 67-72], phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin. Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215-218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.

Effect of troponin-tropomyosin complex and Ca2+ on conformational changes in F-actin induced by myosin subfragment-1.

The interaction of regulated and unregulated actin of myosin-free ghost single fibre with myosin subfragment-1 free of 5,5'-dithiobis(2-nitrobenzoic acid) light chains was investigated by polarized microphotometry. The anisotropy of intrinsic tryptophan fluorescence of regulated actin is Ca2+-dependent and has a maximal value at low (pCa greater than or equal to 7) and a minimal value at high (pCa less than or equal to 6) concentrations of Ca2+. The interaction of myosin subfragment-1 with actin induces cooperative changes in actin structure, which manifest themselves in a decrease in the anisotropy of tryptophan fluorescence. The cooperativity of conformational changes in actin, induced by myosin subfragment-1, is high for regulated actin in the absence of Ca2+ and low for unregulated and regulated actin in the presence of Ca2+. The data obtained suggest that the decrease of the flexibility of actin filaments, induced by tropomyosin or by Ca-unsaturated troponin-tropomyosin complex, results in increased cooperativity of conformational changes of actin induced by myosin subfragment-1.

Detection of conformational changes in actin by proteolytic digestion: Evidence for a new monomeric species

When KCl is added to a solution of G-actin to induce full polymerization, a decrease in the rate at which actin undergoes enzymatic proteolysis occurs. This decrease cannot be accounted for by factors affecting the enzymes employed, but rather appears to be due to a change in the conformation of G-actin. Partially polymerized actin solutions also show a reduction in digestibility which is dependent on the F-actin content, suggesting that F-actin is essentially indigestible. Moreover, low rates of digestion were also observed at sub-critical actin concentrations, where actin in the presence of 0.1 m-KCl does not polymerize. This indicates that a confomational change occurs in G-actin before the polymerization step. At sub-critical concentrations in 0.1 m-KCl, actin is in a truly monomeric state as judged by its viscosity characteristics, its inability to enhance the rate of polymerization of G-actin and its possession of ATP as the actin-bound nucleotide. These data support the existence of a new species of actin, called F-ATP-actin monomer, which has the same physical properties and the same bound nucleotide as G-actin, but digestion characteristics like F-actin. Since F-ATP-actin monomers have the same low susceptibility to proteolysis as F-ADP-actin polymers, and because both G-ATP-actin and G-ADP-actin have similar high rates of digestion, the observed change in the conformation of actin cannot be due to the phosphorylated state of the actin-bound nucleotide. Instead, the conformational change appears to be caused by the addition of KCl to G-actin. The newly-detected monomeric species is considered to be an intermediate in the polymerization process where F-ATP-actin monomers form a population of polymerizable molecules which must reach a critical concentration before nucleation and F-actin polymer formation begin.

Conformational change and cooperativity in actin filaments free of tropomyosin.

The decrease in amplitude of the electron spin resonance spectrum of the cysteine-bound spin-label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinoxyl, brought about by the magnetic interaction with tightly bound manganous ion, was used as a probe of conformational change in actin on binding myosin. The magnitude of this "spin--spin" interaction first decreased then increased on increasing saturation of the actin filament with heavy meromyosin subfragment-1. That the "spin--spin" interaction occurred between spins of adjacent monomers was demonstrated by the observation that the change in magnitude of the "spin--spin" interaction was maintained on binding of heavy meromyosin subfragment-1 to copolymers in which actin monomers containing both manganous ion and spin label were diluted 7-fold with native actin monomers. These data provide evidence for a conformational change in actin on interacting with heavy meromyosin subfragment-1. Further, the fact that not only the magnitude but also the sense of the change in the "spin--spin" interaction is a function of increasing saturation with heavy meromyosin subfragment-1 indicates that the monomers of the actin filament are capable of cooperative interaction in the absence of tropomyosin.

Bound-nucleotide exchange in actin and actomyosin

Abstract 1. 1. To investigate the possibility that conformational changes in actin are involved in muscle contraction, we have studied the effect of myosin on the exchangeability of the bound nucleotide of F-actin. 2. 2. F-Actin shows a significant incorporation of radioactive nucleotide during incubation with 3H-labeled ATP, and this exchange is greatly enhanced by the interaction of the actin with myosin in superprecipitated actomyosin. 3. 3. In both actin and actomyosin, time-course studies show an initial rapid exchange of about 10% of the bound nucleotide followed by further exchange at a slower steady rate. The amount of rapid initial exchange is identical in actin and actomyosin; no extra rapid burst of exchange accompanies the binding of myosin to the actin or the transformation of the actomyosin gel to the superprecipitated state. Rather, myosin only increases the steady rate of exchange during continued exposure of superprecipitated actomyosin to 3H-labeled ATP.