Anion binding characteristics of the band 3 / 4,4-dibenzamidostilbene-2,2-disulfonate binary complex: evidence for both steric and allosteric interactions.

A novel kinetic approach was used to measure monovalent anion binding to better define the mechanistic basis for competition between stilbenedisulfonates and transportable anions on band 3. An anion-induced acceleration in the release of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) from its complex with band 3 was measured using monovalent anions of various size and relative affinity for the transport site. The K1/2 values for anion binding were determined and correlated with transport site affinity constants obtained from the literature and the dehydrated radius of each anion. The results show that anions with ionic radii of 120-200 pm fall on a well-defined correlation line where the ranking of the K1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate approximately bromide < chloride < fluoride). The K1/2 values for the anions on this line were about 4-fold larger than expected for anion binding to inhibitor-free band 3. Such a lowered affinity can be explained in terms of allosteric site-site interactions, since the K1/2 values decreased with increasing anionic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monovalent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindrance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monovalent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.     

Transient-state kinetic evidence for intersubunit allosteric hysteresis during band 3 anion exchange.

On-line, dual-wavelength stopped-flow spectroscopy was used to follow continuously, the uptake of dithionite (S2O4(2-)) into human erythrocyte resealed ghosts. We describe the general characteristics of a pre-steady-state transient phase measured both with chloride and sulfate as co-anions. We then quantitatively characterize the dithionite concentration dependence of the amplitude factor and relaxation constant (kR) for the transient phase measured in sulfate medium. We also study the dithionite dependence of the steady-state velocity. Our results suggest that dithionite induces a slow conformational change in band 3 leading to hysteresis in the transport velocity. As many as 25 turnovers of the transport cycle per monomer can occur prior to attainment of steady state. Both kR and the amplitude factor for the transient phase were dithionite concentration dependent. In addition, the steady-state velocity showed apparent negative cooperativity. To discriminate between monomeric and intersubunit allosteric hysteresis, we performed a series of critical kinetic tests with cells labeled partially with 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Coverage of 85% of the band 3 monomer population with DIDS caused kR to decrease about 10-fold, the dithionite concentration dependence of kR to change significantly, and the apparent negative cooperativity for the steady-state velocity to be eliminated. These results suggest that intersubunit allosteric hysteresis makes a significant contribution to dithionite transport by band 3.     

Normal band 3-cytoskeletal interactions are maintained on tanktreading erythrocytes.

Normal nonnucleated erythrocytes subjected to continuous hydrodynamic shear exhibit membrane deformation or "tanktreading," a process important for reduction of the bulk viscosity of circulating blood. To characterize the effect of this unique process on the erythrocyte membrane we have measured the lateral diffusion of band 3 during tanktreading. Band 3 is normally constrained through interactions with the spectrin-actin cytoskeleton, therefore, any significant disruption of these interactions would result in alterations in band 3 dynamics. Band 3 of human erythrocytes was labeled with dichlorotriazinyl amino fluorescein. After laser photobleaching of an equatorial stripe, fluorescence images were recorded from cells in the presence or absence of shear. The amplitude of induced nonuniformity in the surface distribution of fluorescence was calculated directly from images of unsheared cells. In shear the bleached line rotated with the tanktreading motion of the cells. The surface integral of fluorescence oscillated with this motion. For this case, the amplitude of photobleaching-induced nonuniformity was defined as the amplitude at the fundamental frequency of fast Fourier transforms in time of the oscillations. Shear stress-induced membrane flow did not interrupt the linkage of band 3 with the erythrocyte cytoskeleton. Diffusion coefficient and mobile fraction (1.5 +/- 0.5 x 10(-10) cm2/s and 54 +/- 11%, respectively) were unaffected by shear. The rate of fluorescence recovery of cells in shear was also similar at the centers and at the edges, where in-plane shear forces are maximal.     

Evidence for the allosteric regulation of bacterial glycogen synthesis in vivo

In stationary-phase cultures of either Escherichia coli W4597(K) or G34 in various nutrient conditions there is a 10-fold range of steady-state rates of glycogen synthesis with an essentially constant steady-state level of ATP, presumably reflecting an essentially constant energy charge. The steady-state level of fructose-1,6-diphosphate in these cultures varies from experiment to experiment as a function of the observed rate of glycogen synthesis. These data were fitted to the Hill equation using an assumed Hill coefficient of 2: a plot of [Fru-P₂]²/rate of glycogen synthesis versus [Fru-P₂]² is linear with a correlation coefficient greater than 0.999, indicating a causal relationship between the concentration of Fru-P₂ and the rate of glycogen synthesis. These data provide further evidence that allosteric effects observed in vitro function in vivo.     

Evidence for multisite ADP-ribosylation of neuronal phosphoprotein B-50/GAP-43

The neuronal phosphoprotein B-50/GAP-43 is associated with neuronal growth and regeneration and is involved in the calcium/CaM and G_o signal transduction systems. In particular, B-50 interacts uniquely with CaM by binding in the absence of Ca²⁺. Previously identified as a major neuronal substrate for protein kinase C, which releases CaM via phosphorylation, B-50 has more recently been shown to be a substrate for endogenous ADP-ribosyltransferases. In the present study, we utilized amino acid modification with iodoacetamide and chemical stability to mercury and neutral hydroxylamine to demonstrate that the predominant site of ADP-ribosylation is Cys 3 and/or Cys 4. Chymotryptic peptide mapping further revealed a second, less labelled site of ribosylation in the C-terminal region. The results also demonstrate that, in contrast to PKC phosphorylation, ADP-ribosylation of B-50 does not mediate CaM binding. Since Cys 3 and Cys 4, by palmitoylation, are important for membrane anchoring, our findings suggest that ADP-ribosylation of B-50 may have a role in directing the intracellular localization of the protein. Hence, ribosylation of B-50 may mediate where B-50 interacts with signal transduction pathways.     

Subunit interactions and the allosteric response in phosphorylase.

The contribution of intersubunit interactions to allosterically induced conformational changes in phosphorylase are considered. Phosphorylase a, Pa (phosphorylated at Ser-14), is significantly in the active (R) conformation, while phosphorylase b, Pb (nonphosphorylated), is predominantly in the inactive (T) conformation. The structure of glucose-inhibited (T) Pa has been determined at 2.5-A resolution and atomic coordinates have been measured. These data have been used to calculate the solvent accessible surface area at the subunit interface and map noncovalent interactions between protomers. The subunit contact involves only 6% of the Pa monomer surface, but withdraws an area of 4,600 A2 from solvent. The contact region is confined to the N-terminal (regulatory) domain of the subunit. Half of the residues involved are among the 70 N-terminal peptides. A total of approximately 100 atoms take part in polar or nonpolar contacts of less than 4.0 A with atoms of the symmetry-related monomer. The contact surface surrounds a central cavity at the core of the interface of sufficient volume to accommodate 150-180 solvent molecules. There are four intersubunit salt bridges. Two of these (Arg 10/Asp 32, Ser-14-P/Arg 43) are interactions between the N-terminus of one protomer with an alpha-helix loop segment near the N-terminus of the symmetry-related molecule. These two are relatively solvent accessible. The remainder (Arg 49/Glu 195, Arg 184/Asp 251) are nearer the interface core and are less accessible. The salt bridges at the N-terminus are surrounded by the polar and nonpolar contacts which may contribute to their stability. Analysis of the difference electron density between the isomorphous Pa and Pb crystal structures reveals that the N-terminal 17 residues of Pb are disordered. Pb thus lacks two intermolecular and one intersubunit (Ser-14-P/Arg 69) salt linkage present in Pa. The absence of these interactions in Pb is manifested in the difference in the free energy of T leads to R activation, which is 4 kcal more than that for Pa. Difference Fourier analysis of the T leads to R transition in substrate-activated crystals of Pa suggests that the 70 N-terminal residues undergo a concerted shift towards the molecular core; salt bridges are probably conserved in the transition. It is proposed that the N-terminus, when "activated" by phosphorylation (via a specific kinase) behaves as an intramolecular "effector" of the R state in phosphorylase and serves as the vehicle of homotropic cooperativity between subunits of the dimer.     

Evidence for alpha-ketoglutarate dehydrogenase monomer activity with the aid of enzyme immobilization

Immobilization of pigeon breast muscle alpha-ketoglutarate dehydrogenase on CNBr-activated Sepharose 4B was carried out. Conditions for dissociation of the dimeric enzyme bound to the carrier via a single subunit were determined. Immobilized enzyme monomers with a specific activity higher than that of the dimer were obtained. The immobilized subunits are capable of reassociating with the soluble ones; this is accompanied by the restoration of the initial amount of the matrix-bound protein and the reconstitution of the activity typical of the immobilized enzyme original preparations.     

Evidence for the existence of a functional helical myocardial band

Characterization of local and global contractile activities in the myocardium is essential for a better understanding of cardiac form and function. The spatial distribution of regions that contribute the most to cardiac function plays an important role in defining the pumping parameters of the myocardium like ejection fraction and dynamic aspects such as twisting and untwisting. In general, myocardium shortening, tangent to the wall, and ventricular wall thickening are important parameters that characterize the regional contribution within the myocardium to the global function of the heart. We have calculated these parameters using myocardium displacement fields, which were captured through the displacement-encoding with stimulated echoes (DENSE) MRI technique in three volunteers. High spatial resolution of the acquired data revealed transmural changes of thickening and tangential shortening with high fidelity in beating hearts. By filtering myocardium regions that showed a tangential shortening index of <0.23, we were able to identify the complete or a portion of a macrostructure composed of connected regions in the form of a helical bundle within the left ventricle mass. In this study, we present a representative case that shows the complete morphology of a helical myocardial band as well as two other cases that present ascending and descending portions of the helical myocardial band. Our observation of a helical functional band based on dynamics is in agreement with diffusion tensor MRI observations and gross dissection studies in the arrested heart.     

Evidence for multisite ligand binding and stretching of filamin by integrin and migfilin

Filamin, a large cytoskeletal adaptor, connects plasma membrane to cytoskeleton by binding to transmembrane receptor integrin and actin. Seven of 24 filamin immunoglobulin repeats have conserved integrin binding sites, of which repeats 19 and 21 were shown to be autoinhibited by their adjacent repeats 18 and 20, respectively. Here we show using nuclear magnetic resonance spectroscopy that the autoinhibition can be relieved by integrin or integrin regulator migfilin. We further demonstrate that repeats 19 and 21 can simultaneously engage ligands. The data suggest that filamin is mechanically stretched by integrin or migfilin via a multisite binding mechanism for regulating cytoskeleton and integrin-mediated cell adhesion.     

Evidence for specificity in lipid-rhodopsin interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.     

1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms

The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.     

Evidence for allosteric transitions in secondary structure induced by superhelical stress

Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the torsion constants (alpha) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the torsion constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, or vice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for activation of tissue factor by an allosteric disulfide bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.     

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding

X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a K(d) of 200 nm. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In [(3)H]NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NECA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.     

Evidence for an arginine residue at the allosteric sites of spinach leaf ADPglucose pyrophosphorylase

The covalent modification of spinach leaf ADPglucose pyrophosphorylase leads to inactivation of both activator-stimulated and -unstimulated activity. Inactivation can be prevented if either the activator 3PGA or the inhibitor Pi are present during the modification. Pi proved to be more effective at protecting the enzyme from inactivation as it afforded 50% protection at 51 µM compared to 50% protection by 405 µM 3PGA. Partial modification of the enzyme using [¹⁴C]-phenylglyoxal leads to a decrease in bothV _max,A _0.5 and a decrease in the ability of the 3PGA to stimulate the enzyme's activity. Modification increased the enzyme's susceptibility to inhibition by Pi and completely abolished the cooperative binding of Pi seen in the unmodified enzyme in the presence of 3PGA. Thus, phenylglyoxal appears to interfere, with the normal allosteric regulation of ADPglucose pyrophosphorylase from spinach leaf. Greater than 90% of the enzyme's activity is lost when 7.2 mol [¹⁴C]-phenylglyoxal are bound per mole of tetramer and this label is present in both the larger and small subunits. In addition, inactivation appears to involve two different arginine residues having different rates of modification.     

Evidence from deuterium nuclear magnetic resonance for the temperature-dependent reversible self-association of erythrocyte band 3 in dimyristoylphosphatidylcholine bilayers

Band 3, isolated from human erythrocytes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) deuterated in the terminal methyl groups of the choline head group. By use of Triton X-100 for selective extraction and purification of band 3 and then cholate for subsequent solubilization with the lipid, a number of reconstituted complexes were produced by exhaustive detergent dialysis with protein:lipid weight ratios of between 0.32:1 and 1.25:1. Electron micrographs of negatively stained complexes showed that this method produced large vesicles of greater than 300-nm diameter. Deuterium nuclear magnetic resonance (NMR) spectra from the choline methyl deuterons in bilayer lipid above the liquid-crystal-gel phase transition temperature were shown to change systematically with increasing concentrations of band 3 in the bilayers. The measured quadrupole splittings, taken as the separation of the turning points in the recorded spectra, decreased from a value of 1.28 kHz for pure lipid to 0.98 kHz for bilayers with a protein:lipid ratio of 1.25:1 at 26 degrees C. At 35 degrees C, a more pronounced decrease in the quadrupole splittings was measured. The data from the complexes with protein:lipid ratios up to 0.7:1 (w/w) obey the mathematical treatment for a rapid two-site exchange between lipids at the protein-lipid interface and the bulk lipid phase. The temperature dependence of the measured quadrupole splitting with respect to the protein:lipid ratio indicates that the amount of lipid at the protein-lipid interface increases with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)