Binding modes of L35 to alpha- and beta-semihemoglobins: structural insights into the inequivalence of alpha- and beta-subunits of hemoglobin

It has been thought for several years that the greatly lowered oxygen affinity, high cooperativity, and heterotropic modulation displayed by tetrameric human hemoglobin (Hb) was an exclusive result of the assembly of high affinity alpha(1)beta(1) dimers into alpha(2)beta(2) tetramers. However, in recent times, it has been shown that alpha- and beta-semihemoglobins, namely alpha(heme)beta(apo) and alpha(apo)beta(heme), which are dimers of Hb characterized by a high affinity for oxygen and lack of cooperativity do respond to effectors such as 2-[4-(3,5-dichlorophenylureido) phenoxy]-2-methylpropionic acid (L35), a bezafibrate (BZF) related compound, by decreasing the ligand affinity to a considerable extent (between 60- and 130-fold). In order to shed some light on the structural basis of this phenomenon, we have developed a binding mode of L35 to semihemoglobins through docking analysis using the program GRID. Molecular modelling studies did identify sites on semihemoglobins where favourable interactions with L35 can occur. We found that the effector binds differently to the two semihemoglobins exhibiting high affinity only for the alpha chain heme pocket. The proposed binding models are consistent with the experimental findings and may be rationalized in terms of different hydrophobic and hydrophilic characteristics between alpha- and beta-heme pockets of Hb.     

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists

Having successfully obtained new CCK(1) ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK(1) receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N-substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK(1) receptor binding affinity (compound 1: IC(50)=197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound (1) (coded VL-0395) a receptor binding hypothesis has been provided.     

Modulating calmodulin binding specificity through computational protein design

We report the computational redesign of the protein-binding interface of calmodulin (CaM), a small, ubiquitous Ca(2+)-binding protein that is known to bind to and regulate a variety of functionally and structurally diverse proteins. The CaM binding interface was optimized to improve binding specificity towards one of its natural targets, smooth muscle myosin light chain kinase (smMLCK). The optimization was performed using optimization of rotamers by iterative techniques (ORBIT), a protein design program that utilizes a physically based force-field and the Dead-End Elimination theorem to compute sequences that are optimal for a given protein scaffold. Starting from the structure of the CaM-smMLCK complex, the program considered 10(22) amino acid residue sequences to obtain the lowest-energy CaM sequence. The resulting eightfold mutant, CaM_8, was constructed and tested for binding to a set of seven CaM target peptides. CaM_8 displayed high binding affinity to the smMLCK peptide (1.3nM), similar to that of the wild-type protein (1.8nM). The affinity of CaM_8 to six other target peptides was reduced, as intended, by 1.5-fold to 86-fold. Hence, CaM_8 exhibited increased binding specificity, preferring the smMLCK peptide to the other targets. Studies of this type may increase our understanding of the origins of binding specificity in protein-ligand complexes and may provide valuable information that can be used in the design of novel protein receptors and/or ligands.     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Design, synthesis, binding, and molecular modeling studies of new potent ligands of cannabinoid receptors

In our ongoing program aimed at the design, synthesis, and biological evaluation of novel cannabinoid receptor ligands derived from olivetol and hexyl-resorcinol, we have designed a structural model for new derivatives on the basis of a previous study. Here we report the synthesis, binding, and molecular modeling studies of new potent compounds with high affinity toward CB(1) and CB(2) receptors. Compounds with amidic 'heads' with alkyloxy chains varying in length from 8 to 12 carbon atoms showed nanomolar affinity for both receptors, depending on the type of aromatic backbone. Two of the new compounds, although not very potent, exhibit selectivity for CB(1) receptors (CB(1)/CB(2)=0.07 and 0.08, respectively). Molecular modeling studies fitted this new class of cannabinoid ligands into a CB(1) receptor model, and the qualitative analysis of the results was in general agreement with the CB(1) affinity constants observed experimentally for these derivatives.     

The Binding of Benzopyranes to Human Serum Albumin. A Structure–Affinity Study

The binding of several benzopyranes to serum albumin was studied by equilibrium dialysis at pH7.4 in a 67mM sodium phosphate buffer at 37°C. The equilibrium data were analyzed using a computer program for curve fitting. The binding isotherm for warfarin, 4-hydroxycoumarin, 4-chromanol, coumarin, 3-acetylcoumarin, and benzoic acid can be described by two stoichiometric dissociation constants. Elimination of the 4-hydroxyl group in the coumarin chemical structures decreases the binding affinity of the compounds on the primary binding site of serum albumin, with 4-chromanol the smallest ligand which binds to seroalbumin with high affinity. Thus, the affinity of 4-benzopyranol and the 4-hydroxybenzopyranones greater than that of benzopyranones. On the other hand, elimination of the 2-oxo group in the benzopyranone chemical structures decreases affinity for the secondary binding site.     

Binding of adenosine 3':5'-monophosphate to plasma membranes of Dictyostelium discoideum amoebae

A plasma membrane preparation from Dictyostelium discoideum amoebae which contains the high affinity cAMP receptor is described. Ligand specificity and the kinetics of cAMP association and dissociation using isolated plasma membranes were similar to those of intact cells. The changes in cAMP binding activity which occur as cells proceed through their aggregation program were also reflected in the membrane preparations. However, neither the low affinity cAMP binding site nor the oscillatory cAMP binding behavior observed on intact cells was detected with the membrane preparations.     

Glucagon receptors on isolated hepatocytes and hepatocyte membrane vesicles. Discrete populations with ligand- and environment-dependent affinities

Analysis of glucagon and deshistidine glucagon binding to isolated canine hepatocytes and to hepatocyte membrane vesicles (formed by budding of hepatocytes in hypotonic medium) reveals two separate populations of hormone binding sites. Mathematical modeling further shows that the high affinity population represents 1% of the total in all four cases. Although calculated dissociation constants for hormone binding range from 0.2 to 400 nM, whether considering glucagon or deshistidine glucagon binding, or binding to the high affinity or low affinity receptor populations, receptor affinity increases 2- to 100-fold in the environment of the membrane vesicle; concomitant with this alteration in receptor affinity, receptor selectivity for the structure of the native hormone decreases 1.5- to 40-fold in hepatocyte-derived vesicles. Consideration of receptor affinity in relation to receptor number suggests that hepatocyte glucagon binding is distributed about equally between high and low affinity receptor populations at typical portal hormone levels. Nevertheless, consideration of receptor binding in relation to biological activity suggests that the activity of glucagon in inhibiting carbohydrate flux into glycogen is attributable to occupancy of the high affinity receptor population.     

Quantitative binding studies of a monoclonal antibody to immobilized protein-A.

Binding constants and column capacities are important factors for evaluating an affinity chromatography system. Scatchard plots based on classical equilibrium binding have been used to demonstrate how association constants and column capacities can be computed from simple binding experiments and a commercial computer program. The analysis has been demonstrated on a monoclonal antibody type IgG-1 Kappa against Serratia marcescens nuclease and a commercial protein-A column, Prosep-A. Additional analyses were performed with the same antibody and other protein-A affinity systems and the different binding constants and column capacities obtained confirmed the value of the analysis for evaluating an affinity system.     

Estrogen receptor binding characteristics of 1,11 beta-ethanoestradiol: effect of a 1,11 beta-bridge on steroidal estrogen

As part of an ongoing program to develop high affinity estrogenic ligands we have synthesized the 11 beta-vinyl, 11 beta-ethyl- and 1,11 beta-ethanoestradiols. Because the 1,11 beta-ethano-estradiol had not been previously reported in the literature, the investigation of its receptor binding characteristics would provide valuable insight into the effect of 1/11 beta-substitution. The data obtained in this study indicate that although significant estrogen receptor affinity is present for the 1,11 beta-ethano derivative, the RBA values, 5-22.4%, were far less than those observed (5-300-fold less) for the corresponding 11 beta-ethyl and 11 beta-vinyl estradiols and less than those for the 1-methyl and 11 beta-methyl estradiols. These results suggest that the orientation that the 11 beta-substituent must occupy is directed away from the A-ring and that substituents in the 1-11 pocket produce a detrimental effect on receptor interactions.     

Analysis of a calcium ion binding system composed of two different sites

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca⁺⁺ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca⁺⁺ binding sites and without such solute were titrated with Ca⁺⁺ using Mx as an indicator of free Ca⁺⁺ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca⁺⁺; the association constants of the high affinity site and low affinity site are 4.3 × 10⁵ M^-1 and 9 × 10³ M^-1, respectively. The advantages and limitations of this methodology are discussed.     

Resolution of steroid binding heterogeneity by Fourier-derived affinity spectrum analysis (FASA)

A new mathematical method of analyzing radioreceptor assay data is presented. When there are many binding classes with different affinities, the probability-density function B(p) is described by the equation B(p) = (integral negative infinity to infinity) q(k)f(p-k)dk, where q(k) is the affinity spectrum (density of a particular binding class as a function of affinity) and f(p-k) is a probability function (probability that dissociation constants will fall between k and p-k, where p is the free ligand concentration). This equation is solved for q(k) and evaluated explicitly by Fourier transformation, namely, q(w) = b(w)/f(w), where w is frequency. Since division by f(w) can amplify and high frequency noise present in the experimental data, a Gaussian smoothing function is introduced thus: qs(w) = q(w)e(-w/W0)2, where W0 is a constant. This produces an affinity spectrum defined as a plot of the number of binding sites, qs(k), versus their respective dissociation constants, k. Using a FORTRAN computer program, we verify this algorithm using simulated data. We also apply the procedure to resolve heterogeneous populations of estrogen binders in human endometrium using [3H]estradiol as ligand. Two estrogen binder classes are revealed with dissociation constants approximately 2.5 natural logarithmic units apart. We identify one high-affinity (Kd = 0.18 nM)-low density (70 pM [or 72 fmol/mg protein]) subpopulation and one low affinity (Kd = 2.5 nM)-high density (101 pM [or 102 fmol/mg protein]) subpopulation of estradiol binders. The management of experimental error, sampling limitations, and nonspecific binding are discussed. This method directly transforms experimental data into an easily interpretable representation without mathematical modeling or statistical procedures.     

Binding analyses between Human PPARgamma-LBD and ligands.

The binding characteristics of a series of PPARgamma ligands (GW9662, GI 262570, cis-parinaric acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), LY171883, indomethacin, linoleic acid, palmitic acid and troglitazone) to human PPARgamma ligand binding domain have been investigated for the first time by using surface plasmon resonance biosensor technology, CD spectroscopy and molecular docking simulation. The surface plasmon resonance biosensor determined equilibrium dissociation constants (KD values) are in agreement with the results reported in the literature measured by other methods, indicating that the surface plasmon resonance biosensor can assume a direct assay method in screening new PPARgamma agonists or antagonists. Conformational changes of PPARgamma caused by the ligand binding were detected by CD determination. It is interesting that the thermal stability of the receptor, reflected by the increase of the transition temperature (T(m)), was enhanced by the binding of the ligands. The increment of the transition temperature (DeltaT(m)) of PPARgamma owing to ligand binding correlated well with the binding affinity. This finding implies that CD could possibly be a complementary technology with which to determine the binding affinities of ligands to PPARgamma. Molecular docking simulation provided reasonable and reliable binding models of the ligands to PPARgamma at the atomic level, which gave a good explanation of the structure-binding affinity relationship for the ligands interacting with PPARgamma. Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by the surface plasmon resonance biosensor, indicating that the docking paradigm used in this study could possibly be employed in virtual screening to discover new PPARgamma ligands, although the docking program cannot accurately predict the absolute ligand-PPARgamma binding affinity.     

A requirement for calcium in the transport of cobalamin across the outer membrane of Escherichia coli

Cobalamin (Cbl) transport across the outer membrane of cells of Escherichia coli consists of high affinity Cbl binding to the btuB protein of the Cbl receptor, followed by the proton motive force- and tonB-dependent release of the Cbl into the periplasmic space. During a search for experimental conditions that would mimic this release in vitro with isolated cell envelope particles, we found that calcium was required for the high affinity Cbl binding, and subsaturating calcium concentrations resulted in the decreased affinity of the Cbl receptor for Cbl. The apparent affinity of the Cbl receptor for calcium (KD, approximately 30 nM at pH 6.6) decreased with decreasing pH, resulting in decreased affinity for Cbl at lower pH values. With suboptimal levels of calcium, Cbl binding was decreased by millimolar levels of magnesium.     

Computational design of calmodulin mutants with up to 900-fold increase in binding specificity

Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca²⁺ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca²⁺/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca²⁺ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity.     

Additive method for the prediction of protein-peptide binding affinity. Application to the MHC class I molecule HLA-A*0201

A method has been developed for prediction of binding affinities between proteins and peptides. We exemplify the method through its application to binding predictions of peptides with affinity to major histocompatibility complex class I molecule HLA-A*0201. The method is named "additive" because it is based on the assumption that the binding affinity of a peptide could be presented as a sum of the contributions of the amino acids at each position and the interactions between them. The amino acid contributions and the contributions of the interactions between adjacent side chains and every second side chain were derived using a partial least squares (PLS) statistical methodology using a training set of 420 experimental IC50 values. The predictive power of the method was assessed using rigorous cross-validation and using an independent test set of 89 peptides. The mean value of the residuals between the experimental and predicted pIC50 values was 0.508 for this test set. The additive method was implemented in a program for rapid T-cell epitope search. It is universal and can be applied to any peptide-protein interaction where binding data is known.     

Structural and computational characterization of the SHV-1 beta-lactamase-beta-lactamase inhibitor protein interface

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.     

DESIGN: computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. I. Homologous and heterologous binding to one or two classes of sites

We have developed a versatile computer program for optimization of ligand binding experiments (e.g., radioreceptor assay system for hormones, drugs, etc.). This optimization algorithm is based on an overall measure of precision of the parameter estimates (D-optimality). The program DESIGN uses an exact mathematical model of the equilibrium ligand binding system with up to two ligands binding to any number of classes of binding sites. The program produces a minimal list of the optimal ligand concentrations for use in the binding experiment. This potentially reduces the time and cost necessary to perform a binding experiment. The program allows comparison of any proposed experimental design with the D-optimal design or with assay protocols in current use. The level of nonspecific binding is regarded as an unknown parameter of the system, along with the affinity constant (Kd) and binding capacity (Bmax). Selected parameters can be fixed at constant values and thereby excluded from the optimization algorithm. Emphasis may be placed on improving the precision of a single parameter or on improving the precision of all the parameters simultaneously. We present optimal designs for several of the more commonly used assay protocols (saturation binding with a single labeled ligand, competition or displacement curve, one or two classes of binding sites), and evaluate the robustness of these designs to changes in parameter values of the underlying models. We also derive the theoretical D-optimal design for the saturation binding experiment with a homogeneous receptor class.     

The binding of Ca2+ to Ca2+-transporting microsomes derived from bovine uterine sarcoplasmic reticulum

An obligatory early step in the transport of calcium across the internal membranes of smooth muscle cells is the binding of calcium to the Ca,Mg-ATPase. The characterization of calcium binding to sarcoplasmic reticulum from smooth muscle has not been reported. Calcium binding to a bovine myometrium preparation was investigated using Scatchard analysis and a computer program utilizing weighted least squares curve fitting and an exact mathematical model of binding. This permitted objective measurement of goodness of fit and showed that best fit was obtained using a two site model. Magnesium did not change the affinity for calcium of the two sites; but reduced the number of low affinity sites to half.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.