Divalent Cation-, Nucleotide-, and Polymerization-Dependent Changes in the Conformation of Subdomain 2 of Actin (original) (raw)
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European Journal of Biochemistry, 1993
Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment. Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39-51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer. The ability of monomeric actin to polymerize into helical filaments is a fundamental property of this protein. It is well established that polymerization is strongly influenced by the kind of divalent cation and nucleotide occupying the single high-affinity site for each of these ligands. Actin containing bound Mg2+ (Mg-actin) has a lower critical concentration and polymerizes faster than actin with bound Ca2+ (Ca-actin). Similarly, actin prepared by procedures commonly in use, which contains ATP at the high-affinity site for nucleotide (ATP-actin), has a lower critical concentration and polymerizes faster than ADP-actin. These differences themselves, as well as a number of other observations, are indicative of nucleotide-dependent and metal-ion-dependent alterations in the monomer conformation (for reviews, see [l, 21). Little information, however, is available on the location of these changes in the actin polypeptide chain. Most extensively examined were the alterations in the environment of Cys374 monitored as changes in the fluorescence of actin labelled at this residue with N-iodoacetyl-K-(5-sulfo-l-naphthyl)
Journal of Biological Chemistry, 1986
Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or M$+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G+G*) which is apparently associated with the initiation of polymerization. A much slower conformational change (G+G', or G*+ G*' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-GI-actin (Le. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-Factin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-GI-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site. As we discussed in greater detail in the accompanying paper (l), actin has one high-affinity divalent cation-binding site (Ka-2.6 nM for Ca2+ and-7.3 nM for Mg") and perhaps 5-9 low-affinity cation-binding sites (KA-0.15 mM for Ca2+ and M g + a n d-10 mM for K+). By all procedures commonly in use, monomeric G-actin is prepared with Ca2+ bound to the high-affinity site. Polymerization in vitro of Ca-G-actin can be induced by addition of sufficient Mg+, Ca2+, or K+ to
Biophysical Journal, 1995
Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2' quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCI2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.
Replacement of ATP with ADP Affects the Dynamic and Conformational Properties of Actin Monomer
Biochemistry, 1999
The effect of the replacement of ATP with ADP on the conformational and dynamic properties of the actin monomer was investigated, by means of electron paramagnetic resonance (EPR) and fluorescence spectroscopic methods. The measurement of the ATP concentration during these experiments provided the opportunity to estimate the time dependence of ADP-Mg-G-actin concentration in the samples. According to the results of the fluorescence resonance energy transfer experiments, the Gln-41 and Cys-374 residues are closer to each other in the ADP-Mg-G-actin than in the ATP-Mg-G-actin. The fluorescence resonance energy transfer efficiency increased simultaneously with the ADP-G-actin concentration and reached its maximum value within 30 min at 20°C. The EPR data indicate the presence of an ADP-Mg-G-actin population that can be characterized by an increased rotational correlation time, which is similar to the one observed in actin filaments, and exists only transiently. We suggest that the conformational transitions, which were reflected by our EPR data, were coupled with the transient appearance of short actin oligomers during the nucleotide exchange. Besides these relatively fast conformational changes, there is a slower conformational transition that could be detected several hours after the initiation of the nucleotide exchange.
Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization
Journal of Biological Chemistry, 1988
Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATPactin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 m M at pH 7.0 Kpi increases with pH, indicating that the HpPO; species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.
European Journal of Biochemistry, 1987
Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization Biochim. Biophys. Acta 791, 57-62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-I as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site.
Polymerization and structure of nucleotide-free actin filaments 1 1 Edited by W. Baumeister
Journal of Molecular Biology, 2000
Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is dif®cult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and $99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA ®laments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from ®lament ends. By electron microscopy of negatively stained specimens, NFA forms long ®laments with a persistence length 1.5 times greater than ADP-actin ®laments. Three-dimensional helical reconstructions of NFA and ADP-actin ®laments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically signi®cant less mass density between the two strands of NFA ®laments. When compared with ADP-actin ®laments, the major difference peak of NFA ®laments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA ®laments is not accessible to free nucleotide in the solution. The af®nity of NFA ®laments for rhodamine phalloidin is lower than that of native actin ®laments, due to a lower association rate. This work con®rms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of ®laments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the ®lament.
Polymerization and structure of nucleotide-free actin filaments1
Journal of Molecular Biology, 2000
Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is dif®cult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and $99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA ®laments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from ®lament ends. By electron microscopy of negatively stained specimens, NFA forms long ®laments with a persistence length 1.5 times greater than ADP-actin ®laments. Three-dimensional helical reconstructions of NFA and ADP-actin ®laments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically signi®cant less mass density between the two strands of NFA ®laments. When compared with ADP-actin ®laments, the major difference peak of NFA ®laments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA ®laments is not accessible to free nucleotide in the solution. The af®nity of NFA ®laments for rhodamine phalloidin is lower than that of native actin ®laments, due to a lower association rate. This work con®rms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of ®laments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the ®lament.
Sequence 18-29 on Actin: Antibody and Spectroscopic Probing of Conformational Changes
Biochemistry, 1994
Experimental evidence for the involvement of the 18-29 site within actin subdomain-1 in the actomyosin weak binding interface includes the inhibition of actomyosin ATPase activity by specific peptide antibodies [Adams, S., & Reisler, E. (1993) Biochemistry 32,5051-50561 and by the Dictyostelium actin mutant D24WD25H [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-21311. In this work, the effect of the 18-29 peptide antibodies on the polymerization and conformation of actin has been characterized. Binding of antibody to the 18-29 site strongly inhibited the MgCl2-induced polymerization of G-actin, had a much weaker impact on the CaC12 polymerization of actin, and showed very little effect on the NaCl polymerization of G-actin. These observations were linked to the binding of the 18-29 antibody to the different forms of actin. In sedimentation assays, the (18-29) IgG bound more strongly to Mg-F-and Mg-G-actins than to Ca-F-and Ca-G-actins, respectively. The binding of IgG to F-actin decreased sharply with an increase in ionic strength. Antibody binding to the 18-29 site induced conformational changes within the nucleotide cleft, both slowing the rate of nucleotide exchange and increasing the fluorescence intensity of actin-bound €ATP. The increased fluorescence of a dansyl probe attached to Gln-41 and a pyrene probe attached to Cys-374 demonstrated that antibody binding also caused local perturbations in the DNase I loop of subdomain-2 and at the C-terminus of actin. These results are discussed in terms of actin plasticity and its implications for actomyosin interactions. ~~~~ ~~~~~ ~~ ~~~ ~ ~ -~ ~ ~~~ monomer (Orlova & Egelman , 1992). The opposite effect, namely, a cooperative stabilization of subdomain-2 and the Abbreviations: S-1, myosin subfragment-1; 18-29 peptide antibodies, affinity-purified IgG and Fab fragments of polyclonal antibodies actin filament, has been observed upon the binding of BeF,, directed against sequence 18-29 (KAGFAGDDAPRAY) from the N-terminus of rabbit a-skeletal actin; ELISA, enzyme-linked immunosorbent assay@); ATP, adenosine 5'-triphosphate; pyrenyl actin, actin labeled at Cys-374 with N-(1-pyreny1)iodoacetamide; 5-IAF actin, actin labeled at Cys-374 with 5-(iodoacetamido)fluorescein; DED actin, actin labeled at Gln4lwith dansylethylenediamine; EDTA, ethylenediaminetetraacetic acid; Mg-G (or F)-actin, actin with Mg2+ bound in the highaffinity cation site; Ca-G (or F)-actin, actin with Ca2+ bound in the high-affinity cation site.