Isothermal titration calorimetric studies on the binding of deoxytrimannoside derivatives with artocarpin: implications for a deep-seated combining site in lectins

The carbohydrate binding specificity of the seed lectin from Artocarpus integrifolia, artocarpin, has been elucidated by the enzyme-linked lectin absorbent assay [Misquith, S., et al (1994) J. Biol. Chem. 269, 30393-30401], wherein it was demonstrated to be a Man/Glc specific lectin with high affinity for the trisaccharide present in the core of all N-linked oligosaccharide chains of glycoproteins. As a consequence of this characterization, the binding epitopes of this trisaccharide, 3, 6-di(alpha-D-mannopyranosyl)-D-mannose, for artocarpin were investigated by isothermal titration calorimetry using its monodeoxy as well as Glc and Gal analogues. The thermodynamic data presented here implicate 2-, 3-, 4-, and 6-hydroxyl groups of the alpha(1-3) Man and alpha(1-6) Man residues, and the 2- and 4-OH groups of the central Man residue, in binding to artocarpin. Nevertheless, alpha(1-3) Man is the primary contributor to the binding affinity, unlike other Man/Glc binding lectins which exhibit a preference for alpha(1-6) Man. In addition, unlike the binding reactions of most lectins reported so far, the interaction of mannotriose involves all of its hydroxyl groups with the combining site of the lectin. Moreover, the free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside estimated from the corresponding monodeoxy analogues show nonlinearity, suggesting differential contributions of the solvent and protein to the thermodynamics of binding of the analogues. Thus, this study not only provides evidence for the extended site recognition of artocarpin for the trimannoside epitope but also suggests that its combining site is best described as a deep cleft as opposed to shallow indentations implicated in other lectins.     

How a plant lectin recognizes high mannose oligosaccharides

The crystal structure of Pterocarpus angolensis seed lectin is presented in complex with a series of high mannose (Man) oligosaccharides ranging from Man-5 to Man-9. Despite that several of the nine Man residues of Man-9 have the potential to bind in the monosaccharide-binding site, all oligomannoses are bound in the same unique way, employing the tetrasaccharide sequence Manalpha(1-2)Manalpha(1-6)[Manalpha(1-3)]Manalpha(1-. Isothermal titration calorimetry titration experiments using Man-5, Man-9, and the Man-9-containing glycoprotein soybean (Glycine max) agglutinin as ligands confirm the monovalence of Man-9 and show a 4-times higher affinity for Man-9 when it is presented to P. angolensis seed lectin in a glycoprotein context.     

Synthesis of fluorine substituted oligosaccharide analogues of monoglucosylated glycan chain, a proposed ligand of lectin-chaperone calreticulin and calnexin

As a part of a exploring the N-glycan-mediated glycoprotein quality control in endoplasmic reticulum, 2-fluorinated derivative Glcalpha1 --> 3Man(F) 1, Glcalpha1 --> 3Man(F)alpha1 --> 2Man2, and Glcalpha1 --> 3Man(F)alpha1 --> 2Manalpha1 --> 2Man 3 were synthesized in a concise manner. These oligosaccharides were subjected to binding studies with calreticulin by using isothermal titration calorimetry. It was revealed that disaccharide 1 was a poor ligand, while tri- (2) and tetrasaccharide (3) had observable affinity.     

Binding specificity of mannose-specific carbohydrate-binding protein from the cell surface of Trypanosoma cruzi

The sugar binding specificity of the recently described mannose-specific carbohydrate-binding proteins (CBP) isolated to homogeneity from both the epimastigote and trypomastigote stages of the pathogenic protozoa Trypanosoma cruzi has been studied by quantitative hapten inhibition of the biotinylated CBPs to immobilized thyroglobulin using model oligosaccharides. The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Manalpha1-2Manalpha1-6Manalpha1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Manalpha1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the DeltaH values to be in good agreement with the van't Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation. In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.     

Crocus sativus lectin recognizes Man3GlcNAc in the N-glycan core structure

Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Manalpha(1-3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Manalpha(1-6)Man branch. Most interestingly, the beta-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn in N-glycans and the l-Fuc linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications.     

Structural basis for the carbohydrate specificities of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity

Artocarpin, a tetrameric lectin of molecular mass 65 kDa, is one of the two lectins extracted from the seeds of jackfruit. The structures of the complexes of artocarpin with mannotriose and mannopentose reported here, together with the structures of artocarpin and its complex with Me-alpha-mannose reported earlier, show that the lectin possesses a deep-seated binding site formed by three loops. The binding site can be considered as composed of two subsites; the primary site and the secondary site. Interactions at the primary site composed of two of the loops involve mainly hydrogen bonds, while those at the secondary site comprising the third loop are primarily van der Waals in nature. Mannotriose in its complex with the lectin interacts through all the three mannopyranosyl residues; mannopentose interacts with the protein using at least three of the five mannose residues. The complexes provide a structural explanation for the carbohydrate specificities of artocarpin. A detailed comparison with the sugar complexes of heltuba, the only other mannose-specific jacalin-like lectin with known three-dimensional structure in sugar-bound form, establishes the role of the sugar-binding loop constituting the secondary site, in conferring different specificities at the oligosaccharide level. This loop is four residues longer in artocarpin than in heltuba, providing an instance where variation in loop length is used as a strategy for generating carbohydrate specificity.     

Lectin KM+-induced neutrophil haptotaxis involves binding to laminin

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.     

Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins

The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.     

Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect"

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal alpha-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, DeltaH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, DeltaH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K(a) values of the multivalent carbohydrates relative to monovalent analogs, known as the "multivalency effect," are shown to be due to more positive entropy (TDeltaS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.     

Thermodynamic characterization of ligand-induced conformational changes in UDP-N-acetylglucosamine enolpyruvyl transferase

The binding of UDP-N-acetylglucosamine (UDPNAG) to the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) was studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDPNAG and also in its absence as demonstrated by MALDI mass spectrometry. The covalent attachment of fosfomycin affects the thermodynamic parameters of UDPNAG binding significantly: In the absence of fosfomycin the binding of UDPNAG is enthalpically driven (DeltaH = -35.5 kJ mol(-1) at 15 degrees C) and opposed by an unfavorable entropy change (DeltaS = -25 J mol(-1) K(-1)). In the presence of covalently attached fosfomycin the binding of UDPNAG is entropically driven (DeltaS = 187 J mol(-1)K(-1) at 15 degrees C) and associated with unfavorable changes in enthalpy (DeltaH = 28.8 kJ mol(-1)). Heat capacities for UDPNAG binding in the absence or presence of fosfomycin were -1.87 and -2.74 kJ mol(-1) K(-1), respectively, indicating that most ( approximately 70%) of the conformational changes take place upon formation of the UDPNAG-MurA binary complex. The major contribution to the heat capacity of ligand binding is thought to be due to changes in the solvent-accessible surface area. However, associated conformational changes, if any, also contribute to the experimentally measured magnitude of the heat capacity. The changes in solvent-accessible surface area were calculated from available 3D structures, yielding a DeltaC(p) of -1.3 kJ mol(-1) K(-1); i.e., the experimentally determined heat capacity exceeds the calculated one. This implies that other thermodynamic factors exert a large influence on the heat capacity of protein-ligand interactions.     

Multithermal titration calorimetry: a rapid method to determine binding heat capacities

Herein a new method that allows binding DeltaCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DeltaH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature re-adjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DeltaCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme-chitotriose complex as a model system.     

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.     

Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.     

A Thermodynamic Characterization of the Binding of Thrombin Inhibitors to Human Thrombin, Combining Biosensor Technology, Stopped-Flow Spectrophotometry, and Microcalorimetry

The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399-575 Da) to human alpha-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K(D) (nM to microM range) at 25 degrees C obtained from the BIACORE analysis correlated well with the inhibition constants K(i) in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K(i) and K(D) values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, DeltaH, and entropy, DeltaS, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in DeltaS for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the DeltaH by 20 kJ mol(-1). DeltaH values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37 degrees C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies.     

Thermodynamics of the binding of the C-terminal repeat domain of Streptococcus sobrinus glucosyltransferase-I to dextran

Glucosyltransferases (GTFs) secreted by mutans streptococci and some other lactic acid bacteria catalyze glucan synthesis from sucrose, and possess a C-terminal glucan-binding domain (GBD) containing homologous, directly repeating units. We prepared a series of C-terminal truncated forms of the GBD of Streptococcus sobrinus GTF-I and studied their binding to dextran by isothermal titration calorimetry. The binding of all truncates was strongly exothermic. Their titration curves were analyzed assuming that the GBD recognizes and binds to a stretch of dextran chain, not to a whole dextran molecule. Both the number of glucose units constituting the dextran stretch (n) and the accompanying enthalpy change (DeltaH degrees ) are proportional to the molecular mass of the GBD truncate, with which the Gibbs energy change calculated by the relation DeltaG degrees = -RT ln K (R, the gas constant; T, the absolute temperature; K, the binding constant of a truncate for a dextran stretch of n glucose units) also increases linearly. For the full-length GBD (508 amino acid residues), n = 33.9, K = 4.88 x 10(7) M-1, and DeltaH degrees = -289 kJ mol-1 at 25 degrees C. These results suggest that identical, independent glucose-binding subsites, each comprising 14 amino acid residues on average, are arranged consecutively from the GBD N-terminus. Thus, the GBD binds tightly to a stretch of dextran chain through the adding up of individually weak subsite/glucose interactions. Furthermore, the entropy change accompanying the GBD/dextran interaction as given by the relation DeltaS degrees = (DeltaG degrees - DeltaH degrees)/T has a very large negative value, probably because of a loss of the conformational freedom of dextran and GBD after binding.     

Thermodynamic analysis of the binding of galactose and poly-N-acetyllactosamine derivatives to human galectin-3.

Galectin-3, with a wide tissue distribution and marked developmental regulation, provides significant insights into the progression of various disease and developmental stages. Recognized by its specificity for galactose, a detailed characterization of its sugar binding ability has been investigated by isothermal titration calorimetry. The results presented here complement well with the earlier studies utilizing hapten inhibition assays. Among the various lactose derivatives studied, A-tetrasaccharide emerged with the highest affinity for binding to galectin-3 combining site. This blood group saccharide exhibited a binding affinity 37-fold higher and a 102 kJ/mol more favorable change in enthalpy over lactose at 280 K indicating the existence of additional subsites for both the alpha1-3-linked N-acetylgalactosamine at the non-reducing end and the alpha1-2-linked L-fucosyl residue. The thermodynamic parameters evaluated for other ligands substantiate further the carbohydrate recognition domain to be part of an extended binding site. Binding thermodynamics of galectin-3 with the galactose derivatives are essentially enthalpically driven and exhibit compensatory changes in DeltaH degrees and TDeltaS owing to solvent reorganization.     

Importance of oligomerisation on Pseudomonas aeruginosaLectin-II binding affinity. In silico and in vitro mutagenesis

The effect of terminal GLY114* deletion on the binding affinity of the PA-IIL lectin toward l-fucose was investigated. Both experimental (isothermal titration calorimetry) and computational (molecular dynamics simulations) methods have shown that the deletion mutation decreases the L-fucose affinity. It implies that the PA-IIL saccharide binding affinity is influenced by the dimerization of the lectin. A detailed analysis of computational data confirms the key role of electrostatic interactions in the PA-IIL/saccharide binding.     

Positive heat capacity change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap

Specific recognition of the mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change (DeltaC(degree)(p) = +1.92 +/- 0.93 kJ.mol(-1).K(-1)), which was confirmed by direct ITC measurements (DeltaC(degree)(p) = +1.941 +/- 0.059 kJ.mol(-1).K(-1)). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive DeltaC(degree)(p), the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap-eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E-mRNA 5' cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic DeltaH(degree)(0) = -72.9 +/- 4.2 kJ.mol(-1) and DeltaS(degree)(0) = -116 +/- 58 J.mol(-1).K(-1) of binding to the less negative resultant values, by DeltaH(degree)(sst) = +9.76 +/- 1.15 kJ.mol(-1) and DeltaS(degree)(sst) = +24.8 +/- 2.1 J.mol(-1).K(-1) (at 293 K), while the corresponding DeltaC(degree)(p)(sst) = -0.0743 +/- 0.0083 kJ.mol(-1).K(-1) is negligible in comparison with the total DeltaC(degree)(p) .     

Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9)

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man(8) and Man(9). Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manalpha(1-2)Manalpha (which represents the terminal disaccharide of all three arms of Man(9)) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manalpha(1-2)Manalpha complex revealed that CVN recognizes the stacked conformation of Manalpha(1-2)Manalpha through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manalpha(1-2)Manalpha(1-2)Man, methyl Manalpha(1-2)Manalpha(1-3)Man and methyl Manalpha(1-2)Manalpha(1-6)Man with remarkable selectivity, and binds these trisaccharides with K(A) values ranging from 8.1x10(3)M(-1) to 6.6x10(6)M(-1). Site 2 is less selective in that it binds all three trisaccharides with similar K(A) values ranging from 1.7 to 3.7(+/-0.3)x10(5)M(-1), but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display alpha(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.     

Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (DeltaH(obs)) and the negative heat capacity change (DeltaC(p,obs)) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined DeltaH(obs) over a much wider temperature range (5-60 degrees C) and as a function of monovalent salt concentration and type for SSB binding to (dT)(70) under solution conditions that favor the fully wrapped (SSB)(65) complex (monovalent salt concentration >or=0.20 M). Over this wider temperature range we observe a strongly temperature-dependent DeltaC(p,obs). The DeltaH(obs) decreases as temperature increases from 5 to 35 degrees C (DeltaC(p,obs) <0) but then increases at higher temperatures up to 60 degrees C (DeltaC(p,obs) >0). Both salt concentration and anion type have large effects on DeltaH(obs) and DeltaC(p,obs). These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 degrees C), the midpoint of which shifts to higher temperature (above 35 degrees C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br(-) > Cl(-) > F(-), and these anions are then released upon binding ssDNA, affecting both DeltaH(obs) and DeltaC(p,obs). We conclude that the experimentally measured values of DeltaC(p,obs) for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.