Cooperative binding of myosin subfragment-1 to the actin-troponin-tropomyosin complex.

The binding of myosin subfragment-1 (S-1) to the F-actin-troponin-tropomyosin complex (regulated F-actin) was examined in the presence of ADP (ionic strength, 0.23 M; 22 degrees C) by using the ultracentrifuge and S-1 blocked at SH1 with iodo[14C]acetamide. S-1 . ADP binds with positive cooperativity to regulated F-actin, both in the presence and absence of calcium; it binds independently to unregulated actin. With and without Ca2+ at very low levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds to the regulated actin with less than 1% of the strength that it binds to unregulated actin, whereas at high levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds about 3-fold more strongly to the regulated actin than it does to unregulated actin. The major difference between the results obtained in the presence and absence of Ca2+ with regulated actin is that, in the absence of Ca2+, the binding of S-1 . ADP remains weak until a higher free S-1 . ADP concentration is reached and the transition to strong binding is much more cooperative. These results are consistent with a model that is basically similar to the cooperative binding model of Hill[Hill, T.L. (1952) J. Chem. Phys. 20, 1259-1273] and of Monod et al. [Monod, J., Wyman, J. & Changeux, J. (1965) J. Mol. Biol. 12, 88-118]: The regulated actin filament can exist in two forms, a weak-binding and a strong-binding form; and Ca2+ and S-1 . ADP, acting as allosteric effectors, shift the equilibrium between the two forms.     

Theoretical model for the cooperative equilibrium binding of myosin subfragment 1 to the actin-troponin-tropomyosin complex

Recent experimental data on the equilibrium binding of myosin subfragment 1 (S-1) to regulated actin filaments in the presence and in the absence of Ca(2+) are analyzed by using a linear Ising model. In the model, each tropomyosin-troponin unit (including seven sites on the actin filament) can be in one of two possible states, which have different intrinsic free energies and different binding constants for S-1. Bound S-1 molecules do not interact with each other. There are nearest-neighbor (pair) interactions between these units that depend on the state of each member of the pair and on the number of Ca(2+) bound to one member of the pair. There are two sources of positive cooperativity in this system: the fact that seven actin sites change state together as part of a single unit; and the existence of attractive nearest-neighbor interactions between units. Parameters in the model are evaluated by fitting the data, both in the presence and in the absence of Ca(2+). Several extensions of this model are discussed.     

Crosslinked myosin subfragment 1: a stable analogue of the subfragment-1.ATP complex.

Myosin subfragment 1 (S-1) with its two reactive cysteine groups crosslinked by N,N'-p-phenylenedimaleimide (pPDM), is shown to be a stable analogue of S-1 X ATP and S-1 X ADP X Pi, the predominant complexes present during the steady-state hydrolysis of ATP by S-1. pPDM-S-1 binds to actin with about twice the affinity of S-1 X ATP or S-1 X ADP X Pi, whereas its affinity is 1/100th of that of S-1 X 5'-adenylyl imidodiphosphate and 1/1,000th of that of S-1 X ADP. pPDM-S-1 is also similar to S-1 X ATP and S-1 X ADP X Pi in that its binding to actin is not inhibited by troponin-tropomyosin. In contrast, the binding of S-1, S-1 X ADP, and S-1 X 5'-adenylyl imidodiphosphate to actin is markedly inhibited by troponin-tropomyosin in the absence of Ca2+ when actin is in large excess over S-1. This suggests that modifying S-1 with pPDM stabilizes a conformation which mimics that induced by the binding of ATP.     

Identification of a region susceptible to proteolysis in myosin subfragment-2.

Comparison of the NH2-terminal sequence of myosin short subfragment-2 (Mr of subunit = 37,000) and long subfragment-2 (Mr of subunit = 59,000) demonstrates that the former represents the NH2-terminal portion of the latter and suggests that the hinge region in myosin rod is in the COOH-terminal portion of the long subfragment-2.     

Flexibility of myosin rod, light meromyosin, and myosin subfragment-2 in solution.

Myosin rod was prepared by papain proteolysis of myosin. The components of rod, light meromyosin (LMM) and subfragment-2 (S-2), were prepared by proteolysis of myosin and rod, respectively, using trypsin treated with tosylphenylalanine chloromethyl ketone. S-2, thus prepared, was of greater molecular weight than obtained previously, so that the combined molecular weights of LMM and S-2 were equal to that of rod, and S-2 contained virtually all of the region of the rod susceptible to trypsin. Electro-optical measurements were made on the three fragments in 2 mM sodium pyrophosphate, pH 9.3 at 3 degrees, over a large range of protein concentrations. Analysis of the relaxation of birefringence, at low protein concentration where there was no aggregation, showed that LMM (relaxation time 13.1 micros) behaves as a rigid cylinder. Rod (relaxation time 41.2 micros) and S-2 (relaxation time 6.0 micros) had relaxation rates that were too fast for rigid molecules of their dimensions, and therefore are not straight rods. This implies that myosin rod is flexible in the S-2 portion, presumably in the region susceptible to proteolysis. The implications of rod flexibility for the mechanism of muscle contraction are discussed.     

Kinetic studies of the cooperative binding of subfragment 1 to regulated actin.

The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated. The binding of SF-1 to pure actin, to actin-tropomyosin (actin-TM), or to actin-tropomyosin-troponin (actin-TM-TN) in the presence of Ca2+ was kinetically the same. In each case, the light-scattering transients were biphasic, suggesting a two-step binding of SF-1 to actin. Binding of SF-1 to regulated actin in the absence of Ca2+ was different from binding in its presence and also varied depending on whether SF-1 or regulated actin was in excess. The kinetic results in the absence of CA2+ are explained by a cooperative binding model, in which the initial binding of SF-1 molecules to open (active) actin sites increases the number of open sites. TN-I labeled with the fluorophore 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3 diazole (TN*) was used to probe the state of the actin-TM-TN complex. Binding of SF-1 or CA2+ to regulated actin (in the absence of Ca2+) decreased the fluorescence of actin-TM-TN* by 30%, suggesting that binding of SF-1 or CA2+ induces a similar change in state. The change in fluorescence of TN* was also used to measure the rate of the transition from the active to the relaxed state in the absence of CA2+, which was 430 sec-1 at 4 degrees C in 0.1 M KCl. The lag prior to association of SF-1 with regulated actin (in the absence of Ca2+) was abolished when three SF-1 molecules were prebound per seven G-actin monomers. Similarly, a titration of actin-TM-TN* (in the absence of Ca2+) with SF-1 or SF-1-ADP showed that most actin sites are open, as measured by the fluorescence change, when the occupancy of actin-TM-TN* by SF-1-ADP or SF-1 is approximately 50%. The evidence shows that partial occupancy of a block of G-actin sites (possibly seven) by SF-1 or SF-1-ADP stabilizes the open (active) conformation.     

Motion of subfragment-1 in myosin and its supramolecular complexes: saturation transfer electron paramagnetic resonance.

Molecular dynamics in spin-labeled muscle proteins was studied with a recently developed electron paramagnetic resonance (EPR) technique, saturation transfer spectroscopy, which is uniquely sensitive to rotational motion in the range of 10(-7)-10(-3) sec. Rotational correlation time (tau2) were determined for a spin label analog of iodoacetamide bound to the subfragment-1 (S-1) region of myosin under a variety of conditions likely to shed light on the molecular mechanism of muscle contraction. Results show that (a) the spin labels are rigidly bound to the isolated S-1 (tau2 = 2 x 10(-7) sec) and can be used to estimate values of tau2 for the S-1 region of the myosin molecule; (b) in solutions of intact myosin, S-1 has considerable mobility relative to the rest of the myosin molecule, the value of tau2 for the S-1 segment of myosin being less than twice that for isolated S-1, while the molecular weights differ by a factor of 4 to 5; (c) in myosin filaments, tau2 increases by a factor of only about 10, suggesting motion of the S-1 regions independent of the backbone of the myosin filament, but slower than that in a single molecule; (d) addition of F-actin to solutions of myosin or S-1 increases tau2 by a factor of nearly 10(3), indicating strong immobilization of S-1 upon binding to actin. Saturation transfer spectroscopy promises to provide an extremely useful tool for the study of the motions of the crossbridges and thin filaments in reconstituted systems and in glycerinated muscle fibers.     

Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1).ATP complex.

In our model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1.ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca2+, S-1.ATP should not turn on the regulated actin filament. In the present study, we tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM X S-1) so that functionally it acts like S-1.ATP, although it does not hydrolyze ATP. We found that, in the absence of Ca2+, neither S-1.ATP nor pPDM X S-1.ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto X S-1). In contrast, in the presence of Ca2+, pPDM X S-1.ATP binding almost completely turns on the regulated acto.S-1 ATPase activity. These results can be explained by our original cooperativity model, with pPDM X S-1.ATP binding only approximately equal to 2-fold more strongly to the turned-on form than to the turned-off form of regulated actin. However, our results are not consistent with our alternative model, which predicts that if pPDM X S-1.ATP binds to actin in the absence of Ca2+ but does not turn on the ATPase activity, then it should also not turn on the ATPase activity in the presence of Ca2+.     

Adenylate deaminase binding to synthetic thick filaments of myosin.

Adenylate deaminase (AMP deaminase; AMP aminohydrolase, EC 3.5.4.6), a tetrameric enzyme found at particularly high concentrations in skeletal muscle, has previously been shown to bind strongly to the subfragment-2-portion of myosin in vitro and to the ends of the A band in vivo. It is shown here that when adenylate deaminase is dialyzed with skeletal myosin during formation of synthetic filaments at pH 7.0 it decorates the filament at 14.3-nm intervals, presumably in the region of exposed backbone between crossbridge levels. Optical diffraction of the aggregates reveals both enhancement of reflections arising from underlying myosin organization and other reflections arising from adenylate deaminase arrangement on the filament surface. Adenylate deaminase can thus be used as a specific label in the study of myosin presence and organization.     

Perturbed equilibrium of myosin binding in airway smooth muscle and its implications in bronchospasm

In asthma, the mechanisms relating airway obstruction, hyperresponsiveness, and inflammation remain rather mysterious. We show here that regulation of airway smooth muscle length corresponds to a dynamically equilibrated steady state, not the static mechanical equilibrium that had been previously assumed. This dynamic steady state requires as an essential feature a continuous supply of external mechanical energy (derived from tidal lung inflations) that acts to perturb the interactions of myosin with actin, drive the molecular state of the system far away from thermodynamic equilibrium, and bias the muscle toward lengthening. This mechanism leads naturally to the suggestion that excessive airway narrowing in asthma may be associated with the destabilization of that dynamic process and its resulting collapse back to static equilibrium. With this collapse the muscle undergoes a phase transition and virtually freezes at its static equilibrium length. This mechanism may help to elucidate several unexplained phenomena including the multifactorial origins of airway hyperresponsiveness, how allergen sensitization leads to airway hyperresponsiveness, how hyperresponsiveness can persist long after airway inflammation is resolved, and the inability in asthma of deep inspirations to relax airway smooth muscle.     

Linkage between ligand binding and the dimer-tetramer equilibrium in the Monod-Wyman-Changeux model of hemoglobin.

G. Weber [(1984) Proc. Natl. Acad. Sci. USA 81, 7098-7102] has inferred that the Monod-Wyman-Changeux (MWC) model for ligand binding by hemoglobin would require (contrary to experimental evidence) that increased ligand binding must promote stabilization of alpha 2 beta 2 tetramers with respect to dissociation into alpha beta dimers. Reexamination of the MWC model, however, in the light of general linkage principles and the specific analysis by G. K. Ackers and M. L. Johnson [(1981) J. Mol. Biol. 147, 559-582] shows that the opposite relation must hold, in agreement with experiment. The T form of the tetramer, with low ligand affinity, must be destabilized and progressively dissociates into the high-affinity dimers, designated D, as ligand binding increases. Each ligand molecule bound shifts the standard Gibbs free energy delta G2T for the D-T equilibrium by approximately 3 kcal/mol in favor of the dimer. Thus, T must exist in (at least) five delta G levels of cooperative free energy as it becomes progressively destabilized by successive binding of ligand molecules. Dissociation of the R tetramer to dimers, in contrast, is independent of the amount of ligand bound, so long as dimers and R-state tetramers possess the same (high) affinity for ligand. While the intrinsic ligand-binding constants of the T and R states (KT and KR) remain unchanged throughout by the postulates of the model, the model should not be regarded as a strictly two-state system in view of the multiple free-energy levels indicated above. The present analysis gives approximate, though not precise, agreement with experimental findings on the dimer-tetramer equilibrium considered by Weber and provides a rationale for interpreting other recent experiments concerning this equilibrium.     

Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry

The mechanism of binding of myosin subfragment-1 (S1) to actin in the absence of nucleotides was studied by a combination of stopped-flow fluorescence and ms time resolution electron microscopy. The fluorescence data were obtained by using pyrene-labeled actin and exhibit a lag phase. This demonstrates the presence of a transient intermediate after the collision complex and before the formation of the stable “rigor” complex. The transient intermediate predominates 2–15 ms after mixing, whereas the rigor complex predominates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixing revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed the “arrowhead” appearance characteristic of rigor. The most likely explanation of the disorder of the transient intermediate is that the binding is through one or more flexible loops on the surfaces of the proteins. The transition from disordered to ordered binding is likely to be part of the force-generating step in muscle.     

Distinctions between the two-state and sequential models for cooperative ligand binding.

The two-state and sequential models for positive cooperativity in ligand binding can produce significantly different theoretical binding curves when presented in a Scatchard plot. The conditions that produce the greatest differences have been examined. The theoretical differences have been used to select the two-state model as the best model for describing the binding of acetylcholine to acetylcholine receptors that have been solubilized by Triton X-100 and sodium cholate.     

Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head.

Polymerizations of skeletal G-actin induced by salt and myosin subfragment 1 (S-1) were suppressed by reaction of G-actin with m-maleimidobenzoyl-N-hydroxysuccinimide ester. The G-actin derivative, containing few intramolecular crosslinks and a free maleimide group, was covalently coupled in solution to the S-1 heavy chain. The resulting complex could no longer bind to F-actin. The SH-1 and SH-2 thiols of S-1 were not involved in the complexation and the covalent link was shown to be exclusively on the 50-kDa segment of the S-1 heavy chain. The specific conjugation of the two proteins followed formation of a reversibly associated pyrophosphate-sensitive binary complex which was characterized by different approaches. Potentially, these complexes may be useful in developing the crystallography of actin-bound S-1.     

Preferential and cooperative binding of histone I to chromosomal mammalian DNA.

There is a strong preferential binding of histone I to lymphocyte DNA as compared to Escherichia coli DNA when large DNA fragments (2 times 10-6 daltons) are used. The binding of histone I to lymphocyte DNA and to E. coli DNA is cooperative. The distribution of preferential binding sites has been investigated on fragmented DNA. Most of the 2 times 10-6 dalton fragments were found to have at least one preferential histone I binding site, whereas most of the 2 times 10-5 dalton fragments have none.     

A quantitative model for the cooperative mechanism of human hemoglobin.

A quantitative model has been developed for the cooperative oxygenation of human hemoglobin. The model correlates the structural and energetic features of ligand-linked subunit interactions within the tetrameric molecule and the coupling of these interactions to the binding of oxygen and Bohr protons. Recent findings are incorporated regarding (i) the sites of regulatory energy change within the tetrameric molecule, (ii) the nature of the Bohr effect for tetramers and dimers, (iii) the fractional Bohr proton release at each stage of oxygenation, (iv) relative probabilities of binding to the alpha and beta chains within the tetramer, and (v) an extensive data base recently obtained on the linked processes of oxygenation, proton binding, and subunit interactions [Chu, A. H., Turner, B. W. & Ackers, G. K. (1984) Biochemistry 23, 604-617]. Least squares minimization was used to evaluate from these data the free energies for the various processes. A special feature of the model lies in the synchronization of Bohr proton release with changes in quaternary structure. This leads to the striking prediction that a major fraction (as much as 30%) of tetramers are in the oxy quaternary structure after the first oxygen is bound. The model provides a rationale for the essential features of regulatory energy control, and it defines several kinds of additional information that are needed for a more complete understanding of the hemoglobin mechanism.     

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.     

Photolabeling evidence for calcium-induced conformational changes at the ATP binding site of scallop myosin.

A change in the conformation of the active site of scallop myosin under the influence of regulatory amounts of Ca2+ has been identified by use of the ADP photoaffinity analog 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP). NANDP, trapped at the active site with Mn2+ and vanadate, photolabeled preferentially Arg-128 of the heavy chain in the absence of added Mg2+ and Ca2+ [Kerwin, B. & Yount, R. (1992) Bioconjugate Chem. 3, 328-336]. However, addition of 2 mM Mg2+ and regulatory amounts of Ca2+ (0.01-1 microM) shifted the predominant labeling to Cys-198 of the heavy chain in a Ca(2+)-dependent manner. This Ca(2+)-dependent change in the photolabeling pattern was absent when the regulatory light chains were removed or when the unregulated head (subfragment 1) was examined under similar conditions. These results demonstrate that both Arg-128 and Cys-198 are part of the purine binding site which undergoes a conformational change in response to Ca2+ binding to the regulatory domain.     

Copper binding to the prion protein: structural implications of four identical cooperative binding sites

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd approximately 6 microM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nepsilon2 and Ndelta1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.