The ligandin (non-substrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site).

Glutathione S -transferases (GSTs) play a pivotal role in the detoxification of foreign chemicals and toxic metabolites. They were originally termed ligandins because of their ability to bind large molecules (molecular masses >400 Da), possibly for storage and transport roles. The location of the ligandin site in mammalian GSTs is still uncertain despite numerous studies in recent years. Here we show by X-ray crystallography that the ligandin binding site in human pi class GST P1-1 occupies part of one of the substrate binding sites. This work has been extended to the determination of a number of enzyme complex crystal structures which show that very large ligands are readily accommodated into this substrate binding site and in all, but one case, causes no significant movement of protein side-chains. Some of these molecules make use of a hitherto undescribed binding site located in a surface pocket of the enzyme. This site is conserved in most, but not all, classes of GSTs suggesting it may play an important functional role.     

A novel binding assay identifies high affinity ligands to the rosiglitazone binding site of mitoNEET

A novel outer mitochondrial membrane protein containing [2Fe-2S] clusters, mitoNEET was first identified through its binding to the anti-diabetic drug pioglitazone. Pioglitazone belongs to a family of drugs that are peroxisome proliferator-activated receptor (PPAR) gamma agonists, collectively known as glitazones. With the lack of pharmacological tools available to fully elucidate mitoNEET's function, we developed a binding assay to probe the glitazone binding site with the aim of developing selective and high affinity compounds. We used multiple thiazolidine-2,4-dione (TZD), 2-thioxothiazolidin-4-one (TTD), and 2-iminothiazolidin-4-one (ITD) compounds to establish several trends to enhance ligand development for the purpose of elucidating mitoNEET function.     

Interactions of bilirubin and other ligands with ligandin.

Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.     

Docking study of ligands into the colchicine binding site of tubulin

Cancer is a major cause of mortality in developed countries, following only cardiovascular diseases. Death of cancerous cells can be achieved by stopping mitosis and the antimitotic class of drugs formed by the spindle poisons can be used for this purpose. Their role is to disorganize the mitotic spindle by targeting its main constituent, the microtubules, themselves made of heterodimers of alpha and beta-tubulin. They disrupt the dynamics of the microtubules either by stabilizing them, as do paclitaxel or epothilones, or destabilizing them, as do colchicine. The binding site of colchicine seems to lie between the two units of the tubulin dimer. Here, we report on the characterization of this site by the docking of a series of reference compounds, and the subsequent docking of ligands prepared in our laboratory.     

Mercapturic acid biosynthesis: the separate identities of glutathione-S-aryl chloride transferase and ligandin

To examine if, as has been suggested, a peculiar proteolytic activity of thymus cell lysates might explain failures to detect immunoglobulin (Ig) biosynthesis by thymus cells, lysates of ¹⁴C leucine labelled mouse myeloma cells were incubated with a 10³ excess of unlabelled mouse thymus or spleen cell lysates, and then submitted to immune precipitation to isolate labelled Ig chains. Analysis of the immune precipitates by SDS polyacrylamide gel electrophoresis followed by radioautography failed to provide evidence for the purported proteolytic activity of the thymus cell lysates. Furthermore, thymus cell suspensions uncontaminated by plasma cells were biosynthetically labelled, then lysed in the presence or absence of Trasylol, an inhibitor of trypsin-like protesses. No labelled Ig chains could be detected under either condition of cell lysis. Evidence is presented that the detection of Ig chains synthesized by thymus cell suspensions might result from the contamination of these suspensions by plasma cells.     

Formyl-CoA transferase encloses the CoA binding site at the interface of an interlocked dimer

Formyl-CoA transferase catalyses transfer of CoA from formate to oxalate in the first step of oxalate degradation by Oxalobacter formigenes, a bacterium present in the intestinal flora which is implicated in oxalate catabolism in mammals. Formyl-CoA transferase is a member of a family of CoA-transferases for which no structural information is available. We now report the three-dimensional structure of O.formigenes formyl-CoA transferase, which reveals a novel fold and a very striking assembly of the homodimer. The subunit is composed of a large and a small domain where residues from both the N- and C-termini of the subunit are part of the large domain. The linkers between the domains give the subunit a circular shape with a hole in the middle. The enzyme monomers are tightly interacting and are interlocked. This fold requires drastic rearrangement of approximately 75 residues at the C-terminus for formation of the dimer. The structure of a complex of formyl-CoA transferase with CoA is also reported and sets the scene for a mechanistic understanding of enzymes of this family of CoA-transferases.     

Aminoanthraquinones as novel ligands at the polyamine binding site on the N-methyl-D-aspartate receptor complex

As part of a drug discovery program using high-throughput radioligand-binding assays, aminoanthraquinones were identified as potential modulators of N-methyl-D-aspartate (NMDA) receptor function. Aminoanthraquinones may represent a novel class of polyamine binding site ligands with a unique pharmacophore and may facilitate the rational design of novel NMDA-receptor modulators.     

Activation of the action of penicillopepsin on leucyl-tyrosyl-amide by a non-substrate peptide and evidence for a conformational change associated with a secondary binding site

The cleavage of L-leucyl-L-tyrosyl amide by penicillopepsin is activated about tenfold by L-leucyl-glycyl-L-leucine. The latter is not a substrate. The activator has no effect on K_M. An activation constant K_A = 2.0 ± 0.6 mM has been calculated. Leucyl-glycyl-leucine also affects four bands of the circular dichroism spectrum of the enzyme. A dissociation constant of 2.4 mM has been calculated from a titration of the ellipticity changes. The results suggest that a conformational change caused by binding of the peptide is responsible for the increased catalytic activity.     

Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABA(A) receptors

A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed on GABA(A) receptor subtypes (alpha(1)beta(3)gamma(2s), alpha(2)beta(3)gamma(2s), alpha(4)beta(3)gamma(2s) and alpha(5)beta(3)gamma(2s)), displaying nanomolar affinities as well as selectivity for alpha1- versus alpha2- and alpha3-containing receptors by a factor of between 14 and 26.     

Small molecule ligands define a binding site on the immune regulatory protein B7.1.

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. These compounds inhibit the binding of B7.1 to both CD28 and CTLA4. Both classes of compounds appear to bind the same site, a relatively small portion of the GFCC'C" face of the N-terminal V-set domain of human B7.1, not present in the homologous B7.2 or even mouse B7.1. This site may represent a rare hot spot for small molecule antagonist design of inhibitors of cell-cell interactions, whose ligands may yield leads for the development of novel immunomodulatory medicines.     

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS)

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.     

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.     

Localization of the binding site on fibrin for the secondary binding site of thrombin

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic binding of ligands to angiotensin-converting enzyme

We have investigated the interaction of ligands in the active site of the angiotensin-converting enzyme from rabbit lung by monitoring the concurrent effects of two inhibitors on enzyme activity. A strong synergism is found in the binding of N-acetyl-L-proline (an analog of the COOH-terminal dipeptide portion of preferred substrates) and acetohydroxamate (a zinc ligand). Analysis of the inhibition data with the Yone-tani-Theorell plot yields an unusually low value of 0.0063 for the interaction constant (alpha). This result indicates that each of the above ligands stimulates the binding of the other by about 150-fold. Similar but often less pronounced synergism is observed for other zinc ligands and with some other N-acyl amino acids. These specific structural requirements suggest that the above effect is associated with an induced-fit mechanism which brings the important zinc atom into a catalytically optimal state only in the presence of certain preferred substrates.     

Conformational changes near the cytochrome P450 active site upon binding of two different ligands

It is shown that a stable nitroxyl radical, 4-cyano-2,2,6,6-tetramethylpiperidine-1-oxyl, forms a complex with cytochrome P4502B4 by analogy with the second type substrates by joining directly to pentacoordinate heme iron. The bound radical is inaccessible to water-soluble paramagnetic ions, which confirms its localization in a hydrophobic pocket near the heme. Benzphetamine and N,N-dimethylaniline, the first-type nonpolar substrates, induce conformational changes of the spin-labeled hemoprotein which are evidently accompanied by an increase in the volume of the pocket resulting in emergence of contact with aqueous phase, and the heme-bound spin label becomes accessible to water-soluble paramagnetics. In this case potassium ferricyanide broadens the spin-labeled cytochrome signal and, as a result, lowers the amplitudes of the spectral components. Similar changes were registered at non-micellar concentrations of nonionic detergent Emulgen 913, whose activating effect on hydroxylation reactions is associated, as we showed previously, with its presence in the CYP2B4 active site simultaneously with substrates.     

Use of binding site neighbor-effect parameters to evaluate the interactions between adjacent ligands on a linear lattice. Effects on ligand-lattice association.

A method using binding site "neighbor-effect" parameters (NEPs) is introduced to evaluate the effects of interaction between adjacent ligands on their binding to an infinite linear lattice. Binding site overlap is also taken into account. This enables the conditional probability approach of McGhee & von Hippel to be extended to more complex situations. The general equation for the isotherm is v/LF = SFKF, where v is the ratio of bound ligands to lattice residues, LF is the free ligand concentration, SF is the fraction of binding sites that are free, and KF is the average association constant of a free site. Solutions are derived for three cases: symmetric ligands, and asymmetric ligands on isotropic or anisotropic lattices. For symmetric ligands there is one NEP, E, which is the ratio of the average binding affinity of a free site if the status of the lattice residue neighboring one end of the site is unspecified (left to chance) to the affinity when this residue is free (holding the other neighbor constant). Thus KF is KE2, where K is the affinity of an isolated site. If a site is n residues long, SF is f ffn-1, where f = 1 - nv is the fraction of residues that are free and ff is the conditional probability that a free residue is bordered on a given side by another free residue. The expression for ff is 1/(1 + x/E), where x is v/f, E is (1 - x + [(1 - x)2 + 4x omega]1/2)/2, and omega is the co-operativity parameter. The binding of asymmetric ligands to an isotropic lattice is described by two NEPs; the last case involves four NEPs and a bound ligand orientation parameter. For each case, the expected length distribution of clusters of bound ligands can be calculated as a function of v. When Scatchard plots with the same intercepts and initial slope are compared, it is found that ligand asymmetry lowers the isotherm (relative to the corresponding symmetric ligand isotherm), whereas lattice anisotrophy raises it.     

Coming to grips with integrin binding to ligands

Integrins are alphabeta heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells. They recognize aspartic-acid- or a glutamic-acid-based sequence motifs in structurally diverse ligands. Integrin recognition of most ligands is divalent cation dependent and conformationally sensitive. In addition to this common property, there is an underlying binding specificity between integrins and ligands for which there has been no structural basis. The recently reported crystal structures of the extracellular segment of an integrin in its unliganded state and in complex with a prototypical Arg-Gly-Asp (RGD) ligand have provided an atomic basis for cation-mediated binding of aspartic-acid-based ligands to integrins. They also serve as a basis for modelling other integrins in complex with larger physiologic ligands. These models provide new insights into the molecular basis for ligand binding specificity in integrins and its regulation by activation-driven tertiary and quaternary changes.