Equilibrium dialysis and circular dichroic analysis of the novobiocin-bovine serum albumin complex.

The binding of the drug novobiocin by bovine serum albumin has been analyzed by Scatchard plots at two temperatures and three ionic strengths. In all cases the nonlinearity of the plots indicates heterogeneity of the combining sites on the protein, and an analysis of the changes observed in the circular dichroic spectra of the bound drug supports this observation. By use of a three-constant model the best fit for all of the parameters is achieved by assuming the initial site on the protein to be unique, the next five sites to be homogeneous, and subsequent binding to be described by a simple distribution between two phases. The temperature variation studies reveal that the first site derives its free energy of binding totally from a favorable change in entropy while the second class of five sites derives their free energy of binding from a favorable enthalpy change. Comparisons are made between the binding of novobiocin by bovine albumin and by human albumin.     

Receptor-induced switch in site-site cooperativity during iron release by transferrin.

Iron removal by PPi from the N- and C-terminal binding sites of both free and receptor-complexed transferrin, when the partner site remains occupied with kinetically inert Co(III), has been studied at pH 7.4 and 5.6, at 25 degrees C. At extracellular pH, 7.4, the C-terminal site of free mixed-metal proteins is slightly more labile than its N-terminal counterpart in releasing iron to 0.05 M PPi. The rate and extent of iron removal are retarded from both sites when transferrins are receptor-bound. At endosomal pH, 5.6, the two sites exhibit greater kinetic heterogeneity in iron release to 0.005 M PPi. The N-terminal site is 6 times more facile in relinquishing iron than the C-terminal site when mixed-metal transferrins are free. However, the two sites are affected oppositely upon binding to the receptor. Iron release from the C-terminal site of receptor-complexed CoN-transferrin-FeC is 4 times faster than that from receptor-free protein. In contrast, iron removal from the N-terminal site of receptor-complexed FeN-transferrin-CoC is slowed by a factor of 2 compared to that from free protein. These results help explain our previous observation of a receptor-induced switch in site lability during iron removal from diferric transferrin at pH 5.6 (Bali & Aisen, 1991). Site-site cooperative interactions between the two sites of doubly-occupied transferrin during iron release are altered upon binding to receptor at pH 5.6. Iron in the otherwise weaker binding site of the N-terminal lobe is stabilized, while iron in the relatively stable binding site of the C-terminal lobe is labilized.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Specific association of the CRP-cyclic AMP complex with the control region of the lactose operon of Escherichia coli K 12: a direct fluorimetric study using DNA fragments of different lengths

Specific-site binding of the cAMP . CRP complex to the control region of the lactose operon of E. coli was measured directly. All of the protein molecules did bind specifically, and the binding constant for the major CRP site was not dependent on the length (62, 219 or 301 base pairs) of the DNA fragments used. Comparing the values of the binding constant measured for the major site and for the weaker "operator" CRP site, and referring to the published "consensus sequence" derived from the known CRP sites in a series of operons, we suggest that two sub-sites support additive contribution to the total binding free energy.     

Contributions of amino acid side chains to the kinetics and thermodynamics of the bivalent binding of protein L to Ig kappa light chain

Protein L is a bacterial surface protein with 4-5 immunoglobulin (Ig)-binding domains (B1-B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its beta-sheet. To verify these sites biochemically and to probe their relative contributions to the protein L-Ig kappa light chain (kappa) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. Gel filtration experiments show that PLW binds kappa both in 1:1 complexes and multivalently, consistent with two binding sites. Covalent dimers of the A20C and V51C mutants of PLW were prepared to eliminate site 1 and site 2 binding, respectively; both the A20C and V51C dimers bind kappa in 1:1 complexes and multivalently, indicating that neither site 1 nor site 2 is solely responsible for kappa binding. The A20R mutant was designed computationally to eliminate site 1 binding while preserving site 2 binding; consistent with this design, the A20R mutant binds kappa in 1:1 complexes but not multivalently. To probe the contributions of amino acid side chains to binding, we prepared 75 point mutants spanning nearly every residue of PLW; BIAcore studies of these mutants revealed two binding-energy "hot spots" consistent with sites 1 and 2. These data indicate that PLW binds kappa at both sites with similar affinities (high nanomolar), with the strongest contributions to the binding energy from Tyr34 (site 2) and Tyr36 (site 1). Compared to other protein-protein complexes, the binding is insensitive to amino acid substitutions at these sites, consistent with the large number of main chain interactions relative to side chain interactions. The strong binding of protein L to Ig kappa light chains of various species may result from the ambidextrous binding of the B1-B5 domains and the unimportance of specific side chain interactions.     

A general formulation of the free energy of interaction in cooperative ligand binding: application to hemoglobin

A general method for calculating the change in free energy of cooperative interaction upon partial or complete liganding of a protein containing an arbitrary number of binding sites is presented. This method is used to estimate the change in free energy of interaction upon oxygenation of hemoglobin under physiological conditions, which is compared to the internal energy change upon binding oxygen to the non-interacting binding site.     

Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding.

Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild-type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity--the free energy coupling, delta delta G, is around -8 kJ.mol-1 (Linse S, et al., 1991, Biochemistry 30: 154-162). The mutant, with the substitution Asn 56-->Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant delta delta G < -6.4 kJ.mol-1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+)1 state of the Asn 56-->Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half-saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k.     

Chromophore organization in the higher-plant photosystem II antenna protein CP26

The chlorophyll a/b-xanthophyll-protein CP26 complex belongs to the Lhc protein family. It binds nine chlorophylls and two xanthophylls per 26.6 kDa polypeptide. Determination of the characteristics of each binding site is needed for the understanding of functional organization of individual proteins belonging to the photosystem II supramolecular complex. The biochemical and spectroscopic features of native CP26 are presented here together with identification of pigment binding and energy transitions in different sites. The analysis has been performed via a new approach using recombinant CP26 complexes in which the chromophore content has been experimentally modified. Data were interpreted on the basis of homology with CP29 and LHCII complexes, for which detailed knowledge is available from mutation analysis. We propose that one additional Chl b is present in CP26 as compared to CP29 and that it is located in site B2. We also found that in CP26 three chlorophyll binding sites are selective for Chl a, one of them being essential for the folding of the pigment-protein complex. Two xanthophyll binding sites were identified, one of which (L1) is essential for protein folding and specifically binds lutein. The second site (L2) has lower selectivity and can bind any of the xanthophyll species present in thylakoids.     

Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease

A method is described for measuring equilibrium constants of DNA-protein interactions using gel chromatography. This technique has been used to study the sequence-specific interaction of the HinfI restriction endonuclease with DNA. HinfI has a monomeric molecular weight of 31000 and exists as a dimer in its active form. The protein binds to supercoiled DNA molecules containing its recognition site with an apparent free energy of -13.9 kcal/mol of sites. This interaction is highly salt sensitive and causes a release of 3.4 ion pairs. The affinity of the nuclease for its recognition site is largely independent of both pH (6.5-8.5) and temperature (7-35 degrees C) and was not affected by variations in the degenerate middle position of the site. Linear DNA fragments containing the HinfI recognition site were bound as tightly as supercoiled molecules. Binding to nonspecific DNA sites or to methylated DNA sites was approximately 6 orders of magnitude weaker. In general, enzyme activity and binding affinity paralleled each other.     

Characterization of calcium binding to spectrins.

Calcium binding to brain and erythrocyte spectrins was studied at physiological ionic strength by a calcium overlay assay and aqueous two-phase partitioning. When the spectrins were immobilized on nylon membranes by slot blotting, the overlay assay showed that even though both spectrins bound 45Ca2+, the brain protein displayed much greater affinity for calcium ions than erythrocyte spectrin did. Since the observed binding was weaker than that displayed by calmodulin under similar conditions, the overlay assay results indicated that the binding must be weaker than 1 microM. The phase partition experiments showed that there are at least two sites for calcium on brain spectrin and that calcium binding to one of these sites is reduced significantly by magnesium ions. From the partition isotherm, the dissociation constants were estimated as 50 microM for the Mg(2+)-independent site and 150 microM for the Mg(2+)-dependent site. The phase partition results also showed that erythrocyte spectrin bound calcium ions at least 1 order of magnitude weaker. By examining calcium binding to slot-blotted synthetic peptides, we identified two binding sites in brain spectrin. One mapped to the second putative calcium binding site (EF-hand) in alpha-spectrin and the other to the 36 amino acid residue long insert in domain 11. In addition, a tryptic fragment derived from the C-terminal of erythrocyte alpha-spectrin, which contained the two postulated EF-hands, also bound calcium. These findings suggest that the calcium signal system may also involve direct binding of calcium to spectrin beside known calcium modulators such as calmodulin and calpain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prediction of ligand-binding sites of proteins by molecular docking calculation for a random ligand library

A new approach to predicting the ligand-binding sites of proteins was developed, using protein-ligand docking computation. In this method, many compounds in a random library are docked onto the whole protein surface. We assumed that the true ligand-binding site would exhibit stronger affinity to the compounds in the random library than the other sites, even if the random library did not include the ligand corresponding to the true binding site. We also assumed that the affinity of the true ligand-binding site would be correlated to the docking scores of the compounds in the random library, if the ligand-binding site was correctly predicted. We call this method the molecular-docking binding-site finding (MolSite) method. The MolSite method was applied to 89 known protein-ligand complex structures extracted from the Protein Data Bank, and it predicted the correct binding sites with about 80-99% accuracy, when only the single top-ranked site was adopted. In addition, the average docking score was weakly correlated to the experimental protein-ligand binding free energy, with a correlation coefficient of 0.44.     

Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference

Essential nucleotide contacts between the SV40 large T (tumor) antigen and binding sites I and II on the SV40 genome have been inferred from in vitro methylation- and ethylation-interference experiments. Each site contains two clusters of guanine residues that reduce the specific binding of T antigen when modified. Methylation at any one of nine guanines within site I or any one of five guanines within site II severely interferes with the interaction of T antigen with each respective site. Methylation at any one of a second group of five guanines within site II results in an appreciably weaker effect on the binding of T antigen. A similar inhibitory effect on binding is observed upon ethylation of adjacent phosphate residues. Although there are significant differences in the nucleotide sequence of the two binding sites, the pattern of protein contacts is strikingly similar between sites I and II. Three-dimensional projection reveals that the guanine contacts within each binding site are localized so that the specific binding interactions are accessible from only one face of the DNA helix.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Correlation between binding rate constants and individual information of E. coli Fis binding sites

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.     

Dynamics and assembly of the cytolethal distending toxin

The cytolethal distending toxin (CDT) is a widespread bacterial toxin that consists of an active subunit CdtB with nuclease activity and two ricin-like lectin domains, CdtA and CdtC, that are involved in the delivery of CdtB into the host cell. The three subunits form a tripartite complex that is required to achieve the fully active holotoxin. In the present study we investigate the assembly and dynamic properties of the CDT holotoxin using molecular dynamics simulations and binding free energy calculations. The results have revealed that CdtB likely adopts a different conformation in the unbound state with a closed DNA binding site. The two characterized structural elements of the aromatic patch and groove on the CdtA and CdtC protein surfaces exhibit high mobility, and free energy calculations show that the heterodimeric complex CdtA-CdtC, as well as the CdtA-CdtB and CdtB-CdtC sub-complexes are less energetically stable as compared to the binding in the tripartite complex. Analysis of the dynamical cross-correlation map reveals information on the correlated motions and long-range interplay among the CDT subunits associated with complex formation. Finally, the estimated binding free energies of subunit interactions are presented, together with the free energy decomposition to determine the contributions of residues for both binding partners, providing insight into the protein-protein interactions in the CDT holotoxin.     

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.     

Studies on the mechanism of electron transport in the bc1-segment of the respiratory chain in yeast. II. The binding of antimycin to mitochondrial particles and the function of two different binding sites.

1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants KD1 less than 4 - 10(-13) M and KD2 = 3 - 10(-9) M, respectively. 2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 - 150 pmol per mg of mitochondrial protein. 3. Antimycin bound to the stronger site mainly inhibits NADH-and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN. 4. Under certain conditions cytochrome b and c1 are dispensible components for antimycin-sensitive electron transport. 5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G., and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71-85).