Showing papers on "Cooperative binding published in 1998"

An Analysis of the Origins of a Cooperative Binding Energy of Dimerization

Abstract: The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

Cooperative modulation by eif4g of eif4e-binding to the mrna 5' cap in yeast involves a site partially shared by p20

Abstract: Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

Disabled-2 (Dab2) is an SH3 domain-binding partner of Grb2.

Abstract: Disabled-2 (Dab2), a mammalian structural homolog of Drosophila Disabled (Dab), is a mitogen-responsive phosphoprotein. It has been speculated to be a negative regulator of growth since its expression is lost in ovarian carcinomas. Dab2 contains a C-terminal proline-rich domain with sequences similar to those found in Sos, a guanine nucleotide exchange factor for Ras. The proline-rich sequences of Sos mediate the interaction of Sos with Grb2, an adaptor protein which coupled tyrosine kinase receptors to Sos. Herein, we have investigated the possibility that Dab2 interacts with Grb2. In experiments of co-immunoprecipitation from BAC1.2F5 macrophage cell lysates, significant quantities of Grb2 were associated with both Sos and Dab2, although Dab2 and Sos were not present in the same complex. Transfection of Dab2 into a Dab2-negative cell line (293 cells) decreased the amount of Grb2 associated with Sos, suggesting that Dab2 competes with Sos for binding to Grb2. Proline-rich peptides corresponding to Dab2 (#661-669) and to Sos (#1146-1161) inhibited the binding of Dab2 to Grb2, but were less effective in disrupting the Grb2-Sos complex. The expressed proline-rich domain of Dab2 (#600-730) bound Grb2, but other regions of Dab2 failed to bind Grb2. Both of the individual SH3 domains of Grb2 bound to Sos (N-terminal SH3 domain >> C-terminal SH3 domain), but binding to Dab2 required the intact Grb2, suggesting cooperative binding using both SH3 domains of Grb2. These data indicate that Dab2 binds to the SH3 domains of Grb2 via its C-terminal proline-rich sequences. Dab2 may modulate growth factor/Ras pathways by competing with Sos for binding to Grb2.

Subtype-selective positive cooperative interactions between brucine analogues and acetylcholine at muscarinic receptors: radioligand binding studies.

Abstract: We studied the interactions of strychnine, brucine, and three of the N-substituted analogues of brucine with [3H]N-methylscopolamine (NMS) and unlabeled acetylcholine at m1-m5 muscarinic receptors using equilibrium and nonequilibrium radioligand binding studies. The results were consistent with a ternary allosteric model in which both the primary and allosteric ligands bind simultaneously to the receptor and modify the affinities of each other. The compounds had Kd values in the submillimolar range, inhibited [3H]NMS dissociation, and showed various patterns of positive, neutral, and negative cooperativity with [3H]NMS and acetylcholine, but there was no predictive relationship between the effects. Acetylcholine affinity was increased approximately 2-fold by brucine at m1 receptors, approximately 3-fold by N-chloromethyl brucine at m3 receptors, and approximately 1.5-fold by brucine-N-oxide at m4 receptors. The existence of neutral cooperativity, in which the compound bound to the receptor but did not modify the affinity of acetylcholine, provides the opportunity for a novel form of drug selectivity that we refer to as absolute subtype selectivity: an agent showing positive or negative cooperativity with the endogenous ligand at one receptor subtype and neutral cooperativity at the other subtypes would exert functional effects at only the one subtype, regardless of the concentration of agent or its affinities for the subtypes. Our results demonstrate the potential for developing allosteric enhancers of acetylcholine affinity at individual subtypes of muscarinic receptor and suggest that minor modification of a compound showing positive, neutral, or low negative cooperativity with acetylcholine may yield compounds with various patterns of cooperativity across the receptor subtypes.

Molecular mechanisms of calmodulin's functional versatility

Abstract: Calmodulin (CaM) is a primary Ca2+-binding protein found in all eukaryotic cells. It couples the intracellular Ca2+ signal to many essential cellular events by binding and regulating the activities of more than 40 different proteins and enzymes in a Ca2+-dependent manner. CaM contains two structurally similar domains connected by a flexible central linker. Each domain of the protein binds two Ca2+ ions with positive cooperativity. The binding of Ca2+ transforms the protein into its active form through a reorientation of the existing helices of the protein. The two helices in each helix-loop-helix Ca2+-binding motif are almost antiparallel in Ca2+-free CaM. The binding of Ca2+ induces concerted helical pair movements and changes the two helices in each Ca2+ binding motif to a nearly perpendicular orientation. These concerted helix pair movements are accompanied by dramatic changes on the molecular surface of the protein. Rather than exhibiting a flat, hydrophilic molecular surface as seen in Ca2+-free CaM,...

Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2.

Abstract: The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Structural evidence for the presence of a secondary calcium binding site in human alpha-lactalbumin.

Abstract: The high-resolution X-ray crystal structure of human α-lactalbumin (at 1.8 A) in the presence of an elevated level of calcium reveals a new secondary calcium binding site, 7.9 A away from the primary calcium binding site known in all α-lactalbumin structures so far. The new calcium binding site is different from the zinc and sulfate binding sites [Ren, J., et al. (1993) J. Biol. Chem. 268, 19292−19298] but shares common features with the manganese binding site as described by Gerkin [Gerkin, T. A. (1984) Biochemistry 23, 4688−4697]. The proximity of the manganese and calcium binding region and the location of the functional site on one side of the charged surface of the α-lactalbumin molecule suggest that these binding sites might play a role in the formation of the lactose synthase complex.

Molecular Basis of P450 Inhibition and Activation: Implications for Drug Development and Drug Therapy

Abstract: Three-dimensional homology models of cytochromes P450 (P450) 2B1 and P450 3A4 have been utilized along with site-directed mutagenesis to elucidate the molecular determinants of substrate specificity. Most of the key residues identified in 2B enzymes fall within five substrate recognition sites (SRSs) and have counterparts in bacterial P450 residues that regulate substrate binding or access. Docking of inhibitors into 2B models has provided a plausible explanation for changes in susceptibility to mechanism-based inactivation that accompany particular amino acid side-chain replacements. These studies provide a basis for predicting drug interactions due to P450 inhibition and for rational inhibitor design. In addition, the location of P450 3A4 residues capable of influencing homotropic stimulation by substrates and heterotropic stimulation by flavonoids has been identified. Steroid hydroxylation by the wild-type enzyme exhibits sigmoidal kinetics, indicative of positive cooperativity. Based on the 3A4 model and single-site mutants, a double mutant in SRS-2 has been constructed that exhibits normal Michaelis-Menten kinetics. Results of modeling and mutagenesis studies suggest that the substrate and effector bind at adjacent sites within a single large cavity in P450 3A4. A thorough understanding of the location and structural requirements of the substrate-binding and effector sites in cytochrome P450 3A4 should prove valuable in rationalizing and predicting interactions among the multitude of drugs and other compounds that bind to the enzyme.

Exploring the Leucine-Proline Binding Pocket of the Src SH3 Domain Using Structure-Based, Split-Pool Synthesis and Affinity-Based Selection

Abstract: Structure-based, split-pool synthesis was used to discover non-peptide binding elements for the leucine-proline binding pocket of the Src SH3 domain. Binding characteristics of the protein pocket were then explored by comparing a series of ligands that contains subtle variants of the parent non-peptide binding structure. Further insights into this receptor -ligand interaction were provided by multidimensional NMR structure determination of one of the non-peptide ligands. The study of small molecules that bind protein receptors can provide insight into the molecular forces that govern receptor- ligand interactions. Important binding requirements are often revealed when molecules with very different structures use similar binding contacts to adhere to the protein receptor. We have chosen SH3 domains to test our ability to discover novel structures that have an affinity for the same or similar protein surfaces. Furthermore, analysis of the dependence of the protein affinity on ligand structure reveals much about the intricacies

DNA-Protein Cooperative Binding through Long-Range Elastic Coupling

Abstract: Cooperativity plays an important role in the action of proteins bound to DNA. A simple, mechanical mechanism for cooperativity, in the form of a tension-mediated interaction between proteins bound to DNA at two different locations is proposed. These proteins are not in direct physical contact. DNA segments intercalating bound proteins are modeled as a Worm-Like Chain, which is free to deform in two dimensions. The tension-controlled protein-protein interaction is the consequence of two effects produced by the protein binding. The first is the introduction of a bend in the host DNA and the second is the modification of the bending modulus of the DNA in the immediate vicinity of the bound protein. The interaction between two bound proteins may be either attractive or repulsive, depending on their relative orientation on the DNA. Applied tension controls both the strength and the range of protein-protein interactions in this model. Properties of the cooperative interaction are discussed, along with experimental implications.

A possible allosteric communication pathway identified through a resonance Raman study of four beta37 mutants of human hemoglobin A.

Abstract: The highly conserved tryptophan at position beta37 occupies a key locus at the hinge region within the alpha1beta2 interface of the mammalian hemoglobins. This residue is thought to play an important role in mediating the heme-heme interaction associated with the cooperative binding of oxygen; however, its explicit function is unclear. In this study, the proximal heme environments of several beta37 mutants of adult human hemoglobin (HbA) are probed using visible (Soret band enhanced) resonance Raman spectroscopy. In the equilibrium deoxy derivatives of these mutants, a systematic variation in proximal strain, as reflected in the iron-proximal histidine (F8) stretching frequency, nu(Fe-His), is seen upon mutation of the beta37 residue. The variation in proximal strain correlates with both the ligand binding rates [Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335] and conformational changes observed at the FG corner through X-ray crystallography [Kavanaugh et al. (1998) Biochemistry 37, 4358-4373]. The results from the deoxy samples indicate a plasticity of the tertiary structure within the T quaternary state. The correlation between the X-ray data and the Raman supports the idea that the proximal strain at the heme within the T state can be modulated by a combination of forces including those arising from the hinge region of the alpha1beta2 interface, from the binding of allosteric effectors, and from the degree of iron displacement from the heme plane. Each of these contributors appears to operate through a shifting of the F helix either away from or toward the FG corner. The Raman spectra obtained from the 10 ns CO photoproduct of the beta37 mutant Hb's indicate that these mutants contain an altered coupling between the R state alpha1beta2 interface and the proximal heme environment. This altered coupling could be due to either dissociation of the ligated mutant tetramers into dimers or the formation of an R state tetramer with significantly weakened hydrogen bonds and van der Waals contacts between the alpha1 and beta2 subunits at the interface. In either case, the results reveal a clear-cut structural basis for the quaternary enhancement effect in which the normal R state quaternary structure produces a higher affinity ligand binding site than that which occurs in the corresponding dimeric form of the protein. The normal R state interface is shown to be important for stabilizing a favorable ligand binding environment that persists long enough after laser photolysis to enhance the geminate rebinding process within the photoproduct. The addition of IHP to the solution of mutant COHb proteins results in photoproduct spectra that are all identical and are consistent with the ligand-bound derivatives having either a T state structure or a very strained and anomalous R state structure.

Noncompetitive inhibition of nicotinic acetylcholine receptors by endogenous molecules.

Abstract: Over the past few decades much effort has been expended elucidating the key domains of the nicotinic acetylcholine receptor (AChR) responsible for agonist binding, ion conduction, and gating. An emerging concept in the receptor field has been to consider the receptor entity as a signal transducer that suffers modulatory control by allosterically acting ligands. Of particular interest are the molecules that inhibit the agonist-evoked ion flux activity in a noncompetitive manner: the so-called noncompetitive inhibitors (NCIs). The actual knowledge on the action of NCIs was obtained by using several drugs from exogenous origin. However, several lines of investigation indicate that the receptor protein can be modulated by endogenous substances other than acetylcholine. In this regard, we outline the progress evidenced on the localization of binding sites for drugs of endogenous origin that have been found to directly interact with the AChR in a noncompetitive fashion. Among them we can quote lipids such as steroids and fatty acids, the neurotransmitter 5-hydroxytryptamine (5-HT) and related compounds, as well as the neuropeptide substance P. We present the available experimental evidence indicating the existence of both luminal (located into the ion channel) and nonluminal (located out of the ion channel) binding sites for endogenous NCIs. Particularly, the binding site for substance P is found in the δM2 domain. In addition, the locus for 5-HT is putatively located in the ion channel close to the serine ring, whereas the binding site for two competitive antagonists of 5-HT receptors (e.g., methysergide and spiperone) is located closer to the external end of the ion channel. Instead, fatty acid and steroid molecules bind to nonluminal sites. More specifically, fatty acids may bind to the annular lipid domain of the AChR or/and to the high-affinity quinacrine site (a NCI from exogenous origin) which is located at a nonannular lipid domain. Additionally, steroids may bind to a site located on the extracellular hydrophilic domain of the AChR or/and at the lipid-protein interface, specifically, at the annular lipid domain and/or close to the nonannular quinacrine binding site. J. Neurosci. Res. 52:369–379, 1998. © 1998 Wiley-Liss, Inc.

Structure changes in hemoglobin upon deletion of C-terminal residues, monitored by resonance Raman spectroscopy.

Abstract: Loss of C-terminal residues in hemoglobin raises oxygen affinity and reduces both cooperativity and the Bohr effect. These functional changes are expected from the loss of C-terminal salt bridges, which are seen crystallographically to stabilize the T quaternary structure. Ultraviolet resonance Raman (UVRR) difference spectroscopy confirms that the strength of the T state contacts is diminished when the C-terminal and also the penultimate residues are removed chemically. Deoxy minus CO difference signals arising from the Trpbeta37-Aspalpha94 and Tyralpha42-Aspbeta99 H bonds at the alpha1 beta2 subunit interface are diminished, and at pH 9, the difference spectra reveal a shift to the R quaternary structure. These effects are small for desHisbeta146 Hb and large for desArgalpha141 Hb, consistent with the order of functional changes. In addition, the H bond between the A and E helices is strengthened by removal of Argalpha141 and is further strengthened when the effector molecule IHP (inositol hexaphosphate) is added to deoxy-desArgalpha141 Hb or when its pH is lowered to 5.8. This effect is attributed to the loss of the C-terminal anchor of the alpha chain H helix, which supports the F and A helices. The beta chain is not as sensitive because it has extra F-H interhelix H bonds. Removal of both Hisbeta146 and Tauyrbeta145 produce UVRR changes which are intermediate between desHisbeta146 and desArgalpha141 Hb, although the functional consequences are greater than for desArgalpha141 Hb. Removal of Tyralpha140 as well as Argalpha141 abolishes cooperative binding as well as the Bohr effect, and the UVRR difference signals are also lost, suggesting that quaternary constraints are removed in both the T and the R states. When the approximately 220 cm-1 iron-histidine stretching vibration of the deoxy-proteins is examined, using Raman excitation in resonance with the heme Soret band, the frequency is observed to diminish toward that of deoxyHb A (215 cm-1) as the pH is lowered and IHP is added and to increase toward a completely relaxed value (223 cm-1) as the pH is raised to 9. The relaxation is in the same order as the functional perturbations: desHisbeta146 < desArgalpha141 < desHisbeta146-Tyrbeta145 < desArgalpha141-Tyralpha140. However, even desArgalpha141-Tyralpha140 Hb shows significant reduction in the Fe-His frequency as IHP is added at low pH. The Fe-His frequency is sensitive to both tertiary and quaternary structure changes and is a global indicator of forces at the heme. The order of affinity changes can be understood on the basis of the number of stabilizing H bonds between the F and H helices. Titration curves of the Fe-His frequency against pH are not sigmoidal, consistent with a multiplicity of contributions to the Bohr effect.

Differential effects of S6 mutations on binding of quinidine and 4-aminopyridine to rat isoform of Kv1.4: common site but different factors in determining blockers' binding affinity.

Abstract: Quinidine and 4AP are two nonspecific K channel blockers. Both block voltage-gated K channels from the intracellular side of the membrane and, in most cases, binding is facilitated by channel activation. However, there are distinct differences between quinidine and 4AP in the time- and voltage-dependencies of drug-channel interaction. To learn about the molecular basis underlying the similarities as well as differences in drug actions between quinidine and 4AP, we used rKv1.4 (rat isoform of Kv1.4) as a model and studied: 1) Is there an overlap between the binding sites of quinidine and 4AP? and 2) What factors are involved in determining the binding affinity and kinetics of drug-channel interaction? Our data show that mutations at a position in the S6 domain of rKv1.4 (position 529) can cause dramatic and often opposite effects on quinidine and 4AP binding. For quinidine, the degree of steric hindrance imposed by side chain at position 529 is an important factor in determining binding affinity. For 4AP, 529 mutations that slow the rate of deactivation reduce binding affinity, probably due to a low binding affinity in the open state. This, in conjunction with the observations that 4AP binding is facilitated by channel activation, suggests that optimal 4AP binding may occur in a transitional state between fully-closed and fully-open states. In addition, hydrophobic interactions between blocker molecules and residues at 529 tend to stabilize the binding of both quinidine and 4AP. Because the S6 amino acid sequences are well conserved among many voltage-gated K channels, our findings have general implications in understanding the structural determinants of quinidine and 4AP binding to different K channels.