What makes a binding site a binding site?

Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site.     

Alcohol binding to liposomes by 2H NMR and radiolabel binding assays: does partitioning describe binding?

Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes.     

Iron binding β-hairpin peptides

Posttranslational modification of tyrosine to 3,4-dihydroxyphenylalanine (dopa) yields a unique functional group in biomolecular systems. Oxidation produces a quinone, which can undergo cross linking while deprotonation is well suited to metal binding. Mussels, tunicates and bacteria chelate iron and other metals with multiple dopa subunits. Solution equilibria between catechols and iron indicate favorable assembly though this interaction has not been studied with highly structured biomolecules, such as peptides, despite their widespread biological applications. Here, a series of β-hairpin peptides are generated. Dopa is involved in an aromatic interaction as the edge position. Despite the presence of the surrounding secondary structure dopa readily undergoes oxidation and cross linking. Formation of bispeptide:iron complexes also occur in the presence of mild to significant aromatic interactions.     

Diacylglycerol kinase γ is a novel anionic phospholipid binding protein with a selective binding preference

There are ten isozymes of diacylglycerol kinase (DGK), and they regulate diverse patho-physiological functions. Here, we investigated the lipid-binding properties of DGK isozymes using protein–lipid overlay and liposome-binding assays. DGKγ showed a strong binding activity compared with other DGK isozymes for phosphatidic acid (PA) among the various glycerophospholipids tested. However, DGKγ failed to interact with DG and lyso-PA. Moreover, the isozyme was capable of binding to ceramide-1-phosphate but not to ceramide or sphingosine-1-phosphate. The isozyme bound more strongly to PA containing unsaturated fatty acid than to PA having only saturated fatty acid. An analysis using a series of deletion mutants of DGKγ revealed that the N-terminal region, which contains a recoverin homology domain and EF-hand motifs, is responsible for the PA binding activity of DGKγ. Taken together, these results indicate that DGKγ is an anionic phospholipid binding protein that preferably interacts with a small highly charged head group that is very close to the glycerol or sphingosine backbone.     

Design, synthesis, structure and binding properties of PDZ binding, cyclic β-finger peptides

Protein interaction domains (PIDs) play a critical role in signal transduction. One PID of great interest is the PDZ domain, a 100 amino-acid-residue domain. Most PDZ domains recognize short, C-terminal peptide motives. In the heterodimer of the nNOS-PDZ domain and the α-syntrophin-PDZ domain, however, one PDZ domain forms a β-finger that binds to the other PDZ domain. We show here that cyclic peptides derived from the β-finger of the nNOS-PDZ domain can bind the syntrophin-PDZ domain in the same manner as the whole domain. The structure of three “finger-peptides” of different size has been determined and the binding investigated using calorimetry and NMR-titration experiments.     

Porphyrin-membrane interactions: binding or partition?

Porphyrins are photodynamic drugs employed in an experimental tumor-treatment modality in which cell membranes are one of the primary drug-action sites. To gain insight into the nature of the interaction of these drugs with those primary sites we have studied the affinity of porphyrins to the lipid moieties of biological membranes, at the molecular level. The association of porphyrins to large unilamellar liposomes, modeling the lipid regions of biological membranes was studied (at equilibrium) for deuteroporphyrin IX and protoporphyrin IX, at neutral pH and 37 degrees C, taking into account porphyrin aggregation. Two thermodynamic approaches were investigated: (i) Simple partition equilibria between the external aqueous phase and the lipid bilayer, for drug monomers and dimers. (ii) Binding equilibria of drug monomers and dimers to the lipid bilayer. Using two types of experimental design and processing the data according to the expectations of both approaches, three different models for the binding (differing in the participation assigned to the dimer) were considered. Our major findings are: (a) The data clearly do not fit with the expectations for simple partition equilibria, nor with binding models assuming direct participation of the dimers. (b) The data fit well with a binding process, in which the membrane binds the porphyrin monomers only, with the dimers participating indirectly through the aqueous dimerization equilibrium. (c) At 37 degrees C and neutral pH, for liposomes composed of phosphatidylcholine/cholesterol at molar ratios of 3:2, we found for both investigated species a binding constant of 2.3 x 10(4) M-1. (d) For each species the binding constant is independent of the initial and final states of drug aggregation in the aqueous phase.     

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe²⁺) ≪ ferric chloride (Fe³⁺) < copper sulfate (Cu²⁺) < zinc sulfate (Zn²⁺) < manganese chloride (Mn²⁺) < calcium chloride (Ca²⁺) < magnesium sulfate (Mg²⁺). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe²⁺ compared with any other metal ions, including Fe³⁺.     

Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.     

The binding of inhibitors to α-chymotrypsin

1. The binding of three competitive inhibitors, N-acetyl-d-tryptophan, N-acetyl-l-tryptophan and N-acetyl-d-tryptophan amide, to alpha-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. 2. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. 3. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK(a)7.3. 4. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH7.3. 5. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.     

Co2+ binding to α-lactalbumin

α-Lactalbumin possesses multiple Zn²⁺ binding sites, with the strongest site having an affinity constant of 5×10⁵ M^?1 [Permyakovet al. (1991),J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected in a shift of the thermal denaturation transition temperature by more than 40°. The present work examines Co²⁺ binding to bovineα-lactalbumin, where for this analog of Zn²⁺, multiple binding sites were also found from spectrofluorimetric titrations. The strong site Co²⁺ binding constant was 1.3×10⁶ M^?1. However, in contrast to Zn²⁺ binding, Co²⁺ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluroescence energy transfer measurements between Tb³⁺ in the strong calcium site to Co²⁺ in the strong Zn²⁺ site gave a distance in the range of 14–18 Å, which was in excellent agreement with recent crystallographic data for humanα-lactalbumin [Renet al. (1993), J. Biol. Chem.268, 19292–19298] However, the X-ray structure did not identify the additional zinc sites found from earlier solution studies, presumably due to restrictive crystal packing interactions. The results from the current work confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Presence of a highly efficient “binding” to bacterial contamination can distort data from binding studies

[³H]GABA at low concentrations (5–10 nM) was bound by what appeared to be a GABA receptor binding site in bacterial contamination originating from a batch of distilled water. Under experimental conditions similar to those usually employed in [³H]GABA binding studies, the apparent binding displayed a very high specific component and a high efficiency in terms of [³H]GABA bound per mg of protein. The binding was blocked by muscimol but not by isoguvacine, SR95531 and nipecotic acid. These characteristics suggest that the presence of such spurious binding in the experiments using³H-labeled ligands in brain homogenates may not always be very obvious and, morover, it can result in subtle, but serious, distortions of data from such studies, which may not be immediately recognized.     

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.