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 allosteric effects on p53 oligomerization induced by phosphorylation

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)‐binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the “activating” C‐terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked‐immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild‐type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the “unfolded” conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.     

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 allosteric inhibition sites in the glucose carrier of erythrocytes

2,4-Fluorodinitrobenzene and 2,3-butanedione, which irreversibly inactivate the glucose transfer system of erythrocytes, have been used as probes to determine whether the substrate site and inner and outer sites for reversible inhibitors are located in the same or different regions of the carrier. Inhibitors bound at an inhibition site exposed in the inward-facing but not the outwardfacing form of the carrier (cytochalasin B, androstendione and androstandione) protect the transport system against inactivation by 2,4-fluorodinitrobenzene. Inhibitors bound at an external inhibition site (phloretin) and substrates bound at the transfer site do not protect. In contrast inactivation by 2,3-butanedione is slightly accelerated by internally bound inhibitors, while substrates and substrate analogs bound at the transfer site protect the system. It is shown that fluorodinitrobenzene reacts in the inner inhibition site and butanedione in the substrate site; and further that these sites may be separate binding areas in the carrier linked by allosteric interaction. The consequence of this linkage is that binding of a ligand at the substrate site precludes binding of another ligand at the internal or external inhibition site.     

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 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.     

Protein-protein interactions in the allosteric regulation of protein kinases

Protein-protein interactions involving the catalytic domain of protein kinases are likely to be generally important in the regulation of signal transduction pathways, but are rather sparsely represented in crystal structures. Recently determined structures of the kinase domains of the mitogen-activated protein kinase Fus3, the RNA-dependent kinase PKR, the epidermal growth factor receptor and Ca(2+)/calmodulin-dependent protein kinase II have revealed unexpected and distinct mechanisms by which interactions with the catalytic domain can modulate kinase activity.     

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.     

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.     

Evidence for an allosteric control of the pentose phosphate pathway in Candida utilis

Glucose 6-phosphate dehydrogenase from $\text{Candida utilis}$ exists in two interconvertible species having molecular weights of 110,000 and 220,000, respectively. By use of sodium dodecyl-sulfate the enzyme can be dissociated into 2 electrophoretically separable subunits. The native enzyme was shown to be strongly inhibited by low concentrations of erythrose 4-phosphate and glyceraldehyde 3-phosphate, respectively. Since both these sugar phosphates are metabolites in the later course of the oxidative pathway this regulation may be considered as a negative feedback control of the pentose phosphate cycle.     

Potassium transport in Neurospora. Evidence for a multisite carrier at high pH

At low extracellular pH (4–6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K]_o. At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0°C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K]_o; the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH.     

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.