Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

A comparison of the alkali light chain subunits of vertebrate skeletal muscle myosin in free and heavy chain associated states

The conformations of the alkali light-chain subunits A1 and A2 of vertebrate fast-twitch muscle myosin have been compared for these chains both in their free state and their heavy-chain-associated states by examining the fluorescence parameters of the extrinsic probe 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid attached covalently to the two light chains. The effect of temperature, salt concentration, and ligands such as Mg²⁺ ions, MgADP, and MgATP has also been investigated. In spite of the extensive sequence homology between the two light chains the data indicate that in their free states the fluorophore in the A2 chain resides in a considerably higher hydrophobic environment. It was also found that the presence of the bulky fluorophore on these light chains does not adversely affect their ability to hybridize with Subfragment 1 heavy chains to form ATPase active hybrids. This association to the heavy chains is accompanied by significant changes in the quantum yields of the 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid label indicating that conformational changes do occur during this transition. Mg²⁺ ions were found to cause either an enhancement or a decrease in fluorescence intensity depending on whether the alkali light chains were free or combined to the heavy chains, respectively. Fluorescence perturbation by nucleotide was only observed for the heavy-chain-associated state.     

Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.     

Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosins using electron paramagnetic resonance spectroscopy.

The binding of Ca²⁺, Mg²⁺ and Mn²⁺ to myosins from rabbit skeletal muscle, scallop striated adductor muscle and clam adductor muscle has been investigated. All three myosins bind two moles of divalent metal ion non-specifically and with high affinity (Mn²⁺ > Ca²⁺ > Mg²⁺). In addition, the molluscan myosins bind about a further two moles of Ca²⁺ specifically. Although rabbit myosin binds some Ca²⁺ in the presence of an excess of free Mg²⁺, this binding occurs at the nonspecific sites and should not be taken as evidence for a myosin-linked regulatory system of the type found in molluscan muscles. If such a system exists in vertebrate skeletal muscle, the homologous Ca²⁺-specific sites must be lost during the early stages of the myosin preparation.The characteristic electron paramagnetic resonance spectrum of the bound Mn²⁺ was utilized to confirm the homology of the non-specific sites in vertebrate and molluscan myosins. The sites are located on the “regulatory” class of light chain. Mn²⁺ bound to scallop myosin has a broad electron paramagnetic resonance spectrum, in contrast to the well-resolved spectra that it gives when bound to many other myosin species. This situation was exploited to identify homologous nonspecific, divalent metal-ion sites on the regulatory light chains from a variety of muscle types, including frog skeletal, rabbit cardiac, chicken gizzard and molluscan adductor muscles. When these light chains are combined with desensitized scallop myofibrils the electron paramagnetic resonance spectra of Mn²⁺ bound to the resultant hybrids are dominated by the signal from the non-specific site of the foreign regulatory light chain.     

Identification of the divalent metal ion binding domain of myosin regulatory light chains using spin-labelling techniques

By the criterion of their primary structure myosin regulatory light chains belong to the ‘calcium binding protein’ family and are thought to contain domains related to the E-F hand structure found in parvalbumin. However, the presence of deletions and non-conservative substitutions in the regulatory light chains indicates that, of the four domains apparent in their structure, only the first is competent to bind Ca²⁺ or other divalent metal ions. Electron paramagnetic resonance studies were performed in an attempt to provide experimental verification of this hypothesis. The approach is based on the finding that the paramagnetic Mn²⁺ ion substitutes for Ca²⁺ at the divalent metal ion site and that different regulatory light-chain isotypes contain cysteine residues in different domains which may be spin-labelled with a nitroxide derivative. The electron spin interaction between these two paramagnetic centres is a function of the distance of their separation. Clam (Mercenaria mercenaria) regulatory light chain contains a single cysteine residue located near the first domain and, when spin-labelled, the intensity of the nitroxide signal is reduced by 25% on binding one mole of Mn²⁺. Rabbit skeletal regulatory light chain contains two cysteine residues located in the third and fourth domains and no (<5%) interaction is observed when Mn²⁺ binds to spin-labelled derivatives. Qualitatively, these results suggest that domain 1 is the most likely candidate for the Mn²⁺ binding site. A more quantitative evaluation using the Leigh (1970) theory for the dipolar coupling between rigid-lattice electron spins and various models for the regulatory light chain tertiary structure, including that predicted by Kretsinger &; Barry (1975) for the possibly isologous troponin C structure, substantiates this conclusion.     

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

N-methyl-D-aspartate receptor subunits are non-myosin targets of myosin regulatory light chain

Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the "GXXXR" portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.     

Evidence for the location of divalent cation binding sites on the chloroplast membrane

Summary We have used a combination of chemical labeling and detergent fractionation techniques to locate the divalent cation binding sites on the chloroplast membrane. We determined the Ca²⁺-binding properties of Triton X-100 subchloroplast particles. Photosystem II (TSFII) particles showed one binding site withn=8.4 moles-mg chl^–1 andk _d =20 m. Photosystem I (TSFI) particles contained two binding sites. The first had ann=1.5 moles-mg chl^–1 andk _d =4 m. The second had ann=9.6 moles-mg chl^–1 andk _d =160 m. We have previously shown (Prochaska & Gross,Biochim. Biophys. Acta 376:126, 1975) that the divalent cation binding sites could be blocked using a water-soluble carbodiimide plus a nucleophile. Chlorophylla fluorescence and lightscattering changes were affected at the same carbodiimide concentrations emphasizing the relationship between these processes. The carbodiimide-sensitive sites were found to be located on the Photosystem II particles. A direct correlation between the inhibition of calcium binding and the carbodiimide-mediated incorporation of a (¹⁴C)-nucleophile was observed upon varying such parameters as carbodiimide concentration, nucleophile concentration, pH, and time of reaction. The presence of CaCl₂ during the carbodiimide plus nucleophile modification procedure decreased the incorporation of (¹⁴C)-nucleophile, emphasizing the competition of the CaCl₂ and the modification reagents for some of the same sites. Sodium dodecylsulfate gel electrophoresis of chlorophyll protein aggregates suggested that the site of competition of the calcium chloride and the modification reagents was the light-harvesting chlorophylla/b protein.     

Selective binding of L-thyroxine by myosin light chain kinase

L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.     

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.     

A comparison of myosin light chain subunits in the atria and ventricles of mammals

An SDS-electrophoretic comparison of atrial and ventricular myosin light chain isotypes was performed in mouse, rat, rabbit, dog, pig, rhesus monkey, baboon, human and cow heart. Light chains 1 and 2 in atria and ventricles differed in all species with the possible exception of the rhesus monkey. Relative migration of atrial and ventricular LC-2 isotypes was similar in all species but LC-1 isotypes varied in relative migration rates suggesting increased primary sequence heterogeneity. Order of migration was VLC-1 less than ALC-1 less than ALC-2 less than VLC-2 in mouse, rat, rabbit, dog, baboon and cow and ALC-1 less than VLC-1 less than ALC-2 less than VLC-2 in pig and human heart. No obvious relationship existed between electrophoretic pattern and phylogenetic evolution.     

Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase

The 20,000-dalton light chain of turkey gizzard myosin is phosphorylated at two sites. Dual phosphorylation is observed when both intact myosin and isolated light chains are used as substrates. Phosphorylation of the second site is not observed at higher ionic strength (e.g. 0.35 M KCl). The first phosphorylation site (serine 19) is phosphorylated preferentially to the second site. The latter is phosphorylated more slowly than the first site, and its phosphorylation requires relatively high concentrations of myosin light chain kinase. It is suggested that myosin light chain kinase catalyzes the phosphorylation of both sites on the light chain, and several reasons are cited that make it unlikely that a contaminant kinase is involved. The second phosphorylation site is a threonine residue. Based on the results of limited proteolysis of the light chain, it is concluded that the threonine residue is close to serine 19, and possible locations are threonines 9, 10, and 18. At all concentrations of MgCl2, phosphorylation of the second site markedly increases the actin-activated ATPase activity of myosin and accelerates the superprecipitation response of myosin plus actin.     

Regulatory light chains and scallop myosin. Form of light chain removal or reuptake is dependent on the presence of divalent cations

Readdition of regulatory light chains to regulatory light chain denuded scallop myofibrils, in the presence of magnesium, results in a negatively co-operative restoration of calcium sensitivity as a function of regulatory light chain content. The form of the stoichiometry curves obtained in the presence of 10 mM-EDTA, by light chain removal from scallop myofibrils at various temperatures, are parabolic in shape, consistent with a random removal process. However, in the presence of EDTA at low temperatures, regulatory light chains are removed in a biphasic manner, indicating that the binding constants of the light chains for each myosin head are not equivalent under these conditions. It is shown here that as the temperature is raised, light chain removal by EDTA approaches that of a random process. The stoichiometry curves obtained in the presence of 10 mM-EDTA may therefore be seen as a composite of both a biphasic removal process (temperatures below 20 degrees C) and a random removal process (temperatures above 20 degrees C), there being a temperature-dependent switch in the myosin molecule between 17 and 23 degrees C that governs the mode of light chain removal. These results indicate that both myosin heads must contain light chains for calcium sensitivity and are consistent with our earlier proposals for head-head co-operativity within the scallop myosin molecule.     

Dephosphorylation of distinct sites on the 20 kDa myosin light chain by smooth muscle myosin phosphatase

The dephosphorylation of the myosin light chain kinase and protein kinase C sites on the 20 kDa myosin light chain by myosin phosphatase was investigated. The myosin phosphatase holoenzyme and catalytic subunit, dephosphorylated Ser-19, Thr-18 and Thr-9, but not Ser-1/Ser-2. The role of noncatalytic subunits in myosin phosphatase was to activate the phosphatase activity. For Ser-19 and Thr-18, this was due to a decrease in Km and an increase in k(cat) and for Thr-9 to a decrease in Km. Thus, the distinction between the various sites is a property of the catalytic subunit.     

Characterization and location of divalent cation binding sites in bovine glial fibrillary acidic protein

In our previous work [Yang, Z. W., & Babitch, J. A. (1988) Biochemistry (preceding paper in this issue)] divalent cations were found to be more effective promoters of astroglial filament formation than were monovalent cations. To determine if one or more divalent cation binding sites were the basis for this difference, glial fibrillary acidic protein (GFAP) was attached to nitrocellulose membranes and bathed in 1 microM 45CaCl2 in 60 mM KCl, 0.5 mM MgCl2, and 10 mM imidazole hydrochloride, pH 7.4. After removal of unbound 45Ca2+, GFAP was observed to bind calcium. Flow dialysis experiments showed that GFAP, dissolved in 2 mM Tris-HCl, pH 7.5, contained three classes of binding sites and 0.61 +/- 0.08 (SD), 1.7 +/- 0.4, and 4.6 +/- 0.2 sites per GFAP molecule with dissociation constants of 0.66 +/- 0.01 microM, 6.6 +/- 0.3 microM, and 44 +/- 1 microM, respectively. After addition of 0.5 mM MgSO4 to the flow dialysis solution, the high- and low-affinity sites were not observed while the remaining sites (1.95 +/- 0.15 per GFAP molecule) had a Kd = 2.16 +/- 0.25 microM. This showed that the high- and low-affinity sites are "Ca2+-Mg2+" sites while sites with intermediate affinity are calcium specific. To locate the calcium-binding regions, GFAP peptides were examined for calcium binding by calcium-45 autoradiography. The calcium-specific binding areas were localized in coil I. Computer-assisted analysis of the GFAP sequence revealed several EF-hand-like areas which could be the calcium binding sites. We conclude that divalent cations may play both structural and regulatory roles in astroglial intermediate filaments.     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.