The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

A comparative study of protoheme and heme d catalases : role of the heme and the heme pocket in catalysis and ligand binding

Catalase dismutes H20 2 to O2 and H20. In successive twoelectron reactions H20 2 induces both oxidation and reduction at the heme group. In the first step the protoheme prosthetic group of beef liver catalase forms compound I, in which the heme has been oxidized from Fe3+ to Fe4+=0 and a porphyrin radical has been created. Compound II is formed by the oneelectron reduction of comp I. It retains Fe4+=0 but lacks the porphyrin radical and is catalytically inert. Molecular structures are available for Escherichia coli Hydroperoxidase II, Micrococcus Iysodeiktus, Penicillium vitale and beef liver enzymes, which contain different hemes and heme pockets. In the present work, the pockets and substrate access channels of protoheme (beef liver & Micrococcus) and heme d (HPII of E. coli and Penicillium) catalases have been analysed using Quanta™ and CharmMTM molecular modeling packages on the Silicon Graphics Iris Indigo 2 computer. Experimental studies have been carried out with two catalases, HPII (and its mutants) and beef liver. Fluoride and formate' are inhibitors of both enzymes, and their binding is modulated by the heme and by distal residues N201 & H128. Both HPII and beef liver enzymes form compound I with H202 or peracetate. The reduction of beef liver enzyme compound I to II and the decay of compound II are accelerated by fluoride. The decay of compound II is also accelerated by formate, and this reagent acts as a 2-electron donor towards compound I of both enzymes. It is concluded that heme d enzymes (Penicillium and HPII of E. coli) are formed by autocatalytic transformation of protoheme in a modified pocket which contains a characteristic serine residue as well as a partially occluded heme channel. They are less active than protoheme enzymes but also do not form the inactive compound II species. Binding of peroxide as well as fluoride and formate is prevented by mutation of H128 and modulated by mutation of N201.

Analysis of dynamics and mechanism of ligand binding to Artocarpus integrifolia agglutinin. A 13C and 19F NMR study

Binding of 13C-labeled N-acetylgalactosamine (13C-GalNAc) and N-trifluoroacetylgalactosamine (19F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using 13C and 19F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of 13C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the alpha-anomers of 13C-GalNAc and 19F-GalNAc (k+1 = 1.01 x 10(5) M-1.s-1 and 0.698 x 10(5) M-1.s-1, respectively) are in close agreement with those obtained for the corresponding beta-anomers (k+1 = 0.95 x 10(5) M-1.s-1 and 0.65 x 10(5) M-1.s-1, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s-1, respectively, for the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc. A two-step mechanism has been proposed for the interaction of 13C-GalNAc and 19F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.

Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 angstrom resolution) and citrate-bound (Form-II, 1.90 angstrom resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K-m in the order of acetate > propionate > formate. Further, the K-m for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic beta beta beta alpha beta alpha beta alpha topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes. Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

PocketMatch (version 2.0): A parallel algorithm for the detection of structural similarities between protein ligand binding-sites

Knowledge of protein-ligand interactions is essential to understand several biological processes and important for applications ranging from understanding protein function to drug discovery and protein engineering. Here, we describe an algorithm for the comparison of three-dimensional ligand-binding sites in protein structures. A previously described algorithm, PocketMatch (version 1.0) is optimised, expanded, and MPI-enabled for parallel execution. PocketMatch (version 2.0) rapidly quantifies binding-site similarity based on structural descriptors such as residue nature and interatomic distances. Atomic-scale alignments may also be obtained from amino acid residue pairings generated. It allows an end-user to compute database-wide, all-to-all comparisons in a matter of hours. The use of our algorithm on a sample dataset, performance-analysis, and annotated source code is also included.

Identification of a novel ligand binding motif in the transthyretin channel

The design of therapeutic compounds targeting transthyretin (TTR) is challenging due to the low specificity of interaction in the hormone binding site. Such feature is highlighted by the interactions of TTR with diclofenac, a compound with high affinity for TTR, in two dissimilar modes, as evidenced by crystal structure of the complex. We report here structural analysis of the interactions of TTR with two small molecules, 1-amino-5-naphthalene sulfonate (1,5-AmNS) and 1-anilino-8-naphthalene sulfonate (1,8-ANS). Crystal structure of TTR: 1,8-ANS complex reveals a peculiar interaction, through the stacking of the naphthalene ring between the side-chain of Lys15 and Leu17. The sulfonate moiety provides additional interaction with Lys15` and a water-mediated hydrogen bond with Thr119`. The uniqueness of this mode of ligand recognition is corroborated by the crystal structure of TTR in complex with the weak analogue 1,5-AmNS, the binding of which is driven mainly by hydrophobic partition and one electrostatic interaction between the sulfonate group and the Lys15. The ligand binding motif unraveled by 1,8-ANS may open new possibilities to treat TTR amyloid diseases by the elucidation of novel candidates for a more specific pharmacophoric pattern. (C) 2009 Published by Elsevier Ltd.

Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: Implications to tetramer stability and ligand-binding

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis. (C) 2010 Elsevier Inc. All rights reserved.

Coupling Between GABA(A)-R Ligand-Binding Activity and Membrane Organization in beta-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with beta-cyclodextrin (beta-CD). The mere pre-incubation (PI) at 37A degrees C accompanying the beta-CD treatment was an underlying source of perturbations increasing [H-3]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4A degrees C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the beta-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not beta-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with beta-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, beta-CD is not completely eliminated from the system through centrifugation washings. It was concluded that beta-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual beta-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

A fragment-based approach for ligand binding affinity and selectivity for the liver X receptor beta

Selective modulation of liver X receptor beta (LXR beta) has been recognized as an important approach to prevent or reverse the atherosclerotic process. In the present work, we have developed robust conformation-independent fragment-based quantitative structure-activity and structure-selectivity relationship models for a series of quinolines and cinnolines as potent modulators of the two LXR sub-types. The generated models were then used to predict the potency of an external test set and the predicted values were in good agreement with the experimental results, indicating the potential of the models for untested compounds. The final 2D molecular recognition patterns obtained were integrated to 3D structure-based molecular modeling studies to provide useful insights into the chemical and structural determinants for increased LXR beta binding affinity and selectivity. (C) 2011 Elsevier Inc. All rights reserved.

AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites ( Ile(116), Arg(175), Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 ( peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394 - 3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] ( peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.