The purification and characterization of a globular subfragment of Acanthamoeba myosin II that is fully active when cross-linked to F-actin

Acanthamoeba myosin II contains two heavy chains of Mr 185,000 and two pairs of light chains of Mr 17,500 and 17,000. We now report the purification of a globular proteolytic 103-kDa subfragment of myosin II which contained a 68-kDa NH2-terminal segment of the heavy chain and one pair of intact light chains. The myosin II head fragment expressed full Ca2+-ATPase activity but its actin-activated Mg2+-ATPase activity had a Vmax of only 0.07 s-1 compared to 1.9 s-1 (per head) for filaments of native unphosphorylated myosin II. The head fragment had a similar KATPase to that of filaments (5 versus 4 microM) and about 75% of the head fraction could bind to F-actin in the presence of ATP with a Kbinding of 5.6 microM. The Kbinding of the head fragment may be similar to that of individual heads in the native myosin II filaments although the experimentally determined apparent Kbinding for filaments is much lower, 0.3 microM. The head fragment was covalently cross-linked to F-actin in the absence of nucleotide using the zero length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked actin-myosin head complex hydrolyzed MgATP at a rate equivalent to Vmax for the active dephosphorylated native myosin II. These data indicate that the isolated head fragment had intact catalytic and actin-binding domains but that it bound to F-actin in the presence of ATP in a relatively inactive conformation. When covalently cross-linked to F-actin the head fragment was apparently locked into a catalytically fully active conformation.     

The Pyrococcus exosome complex: structural and functional characterization

The exosome is a conserved eukaryotic enzymatic complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the "RNases PH ring" complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring complex corresponds to the core of the exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the exosome. In the presence of aRrp4, the activity of the core complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the exosome in RNA metabolism in Archaea, as was established in Eukarya.     

Interaction between myosin and F-actin. Correlation with actin-binding sites on subfragment-1

F-Actin bindings to subfragment-1 (S-1) and S-1 after limited proteolysis by trypsin (S-1t) were studied in the absence and presence of ATP by means of ultracentrifugation. No significant difference in the affinities for F-actin was observed between S-1 and S-1t in the absence of ATP. In contrast, the affinity for F-actin in the presence of ATP was decreased about 50 times by the limited proteolysis of the S-1 heavy chain. The S-1 whose SH1 and SH2 groups were cross-linked by N,N'-p-phenylenedimaleimide bound F-actin weakly. The affinity for F-actin was similar to that of unmodified S-1 in the presence of ATP and was also decreased markedly by limited proteolysis of the cross-linked S-1. Reciprocals of the dissociation constant of acto-S-1 complex decreased markedly with increase of ionic strength in the presence of ATP, but decreased only slightly at the rigor state. All these results are consistent with our proposal that S-1 has two different actin binding sites, as reported previously (Katoh, T., Imae, S., & Morita, F. (1984) J. Biochem. 95, 447-454). The mechanism of activation of S-1 ATPase by F-actin is discussed.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Binding of myosin subfragment-1 to F-actin.

During a part of the hydrolytic cycle, myosin head (S1) carries no nucleotide and binds strongly to an actin filament forming a rigor bond. At saturating concentration of S1 in rigor, S1 is well known to form 1:1 complex with actin. However, we have provided evidence that under certain conditions S1 could also form a complex with 2 actin monomers in a filament (Andreev, O.A. & Borejdo, J. (1991) Biochem. Biophys. Res. Comm. 177, 350-356). This view was recently challenged by Carlier & Didry (Carlier, M-F. & Didry, D. (1992) Biochem. Biophys. Res. Comm. 183, 970-974) who interpreted our data by suggesting that F-actin underwent a simple depolymerization and implied that, when only actin in the F-form was scored, the real stoichiometry in our experiments was 1:1. We show here that under conditions of our experiments less than 8% of actin was depolymerized. Moreover, we have repeated the experiments in the presence of phalloidin and show that under these conditions too, when S1 was added slowly to a fixed concentration of F-actin, it formed a different complex with F-actin than when it was added quickly. This confirms our original conclusion that S1 can bind actin in two different ways and shows that depolymerization of F-actin is not responsible for this finding.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.     

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.     

Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.     

Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1

We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and alpha-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2 led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 degrees C, pH 8.0. The MBS-F-actin formed activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and catalytic properties of a cross-linked complex between adrenodoxin reductase and adrenodoxin

A stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c.     

Nucleotide-induced states of myosin subfragment 1 cross-linked to actin

Actomyosin interactions and the properties of weakly bound states in carbodiimide-cross-linked complexes of actin and myosin subfragment 1 (S-1) were probed in tryptic digestion, fluorescence, and thiol modification experiments. Limited proteolysis showed that the 50/20K junction on S-1 was protected in cross-linked acto-S-1 from trypsin even under high-salt conditions in the presence of MgADP, MgAMPPNP, and MgPPi (mu = 0.5 M). The same junction was exposed to trypsin by MgATP and MgATP gamma S but mainly on S-1 cross-linked via its 50K fragment to actin. p-Phenylenedimaleimide-bridged S-1, when cross-linked to actin, yielded similar tryptic cleavage patterns to those of cross-linked S-1 in the presence of MgATP. By using p-nitrophenylenemaleimide, it was found that the essential thiols of cross-linked S-1 were exposed to labeling in the presence of MgATP and MgATP gamma S in a state-specific manner. In contrast to this, the reactive thiols were protected from modification in the presence of MgADP, MgAMPPNP, and MgPPi at mu = 0.5 M. These modifications were compared with similar reactions on isolated S-1. Experiments with pyrene-actin cross-linked to S-1 showed enhancement of fluorescence intensity upon additions of MgATP and MgATP gamma S, indicating the release of the pyrene probe on actin from the sphere of S-1 influence. The results of this study contrast the "open" structure of weakly bound actomyosin states to the "tight" conformation of rigor complexes.     

Integrated mass spectrometry strategy for functional protein complex discovery and structural characterization

The discovery of functional protein complex and the interrogation of the complex structure-function relationship (SFR) play crucial roles in the understanding and intervention of biological processes. Affinity purification-mass spectrometry (AP-MS) has been proved as a powerful tool in the discovery of protein complexes. However, validation of these novel protein complexes as well as elucidation of their molecular interaction mechanisms are still challenging. Recently, native top-down MS (nTDMS) is rapidly developed for the structural analysis of protein complexes. In this review, we discuss the integration of AP-MS and nTDMS in the discovery and structural characterization of functional protein complexes. Further, we think the emerging artificial intelligence (AI)-based protein structure prediction is highly complementary to nTDMS and can promote each other. We expect the hybridization of integrated structural MS with AI prediction to be a powerful workflow in the discovery and SFR investigation of functional protein complexes.     

Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting

Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex.