Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II

BACKGROUND: Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. RESULTS: The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). CONCLUSIONS: The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes.     

Thermodynamic Analysis of Chitooligosaccharide Binding to Urtica dioica agglutinin by Isothermal Titration Calorimetry

UDA (Urtica dioica agglutinin) contains two hevein like domains with two non-identical interacting sites and is specific for chitooligosaccharides. The binding of chitooligosaccharides to UDA was studied by Isothermal Titration Calorimetry. Each site is composed of three subsites, each binding to a sugar residue. Thermodynamic parameters obtained show that while chitobiose has two independent non-interacting sites, chitotriose, chitotetrose and chitopentose have two interacting sites on each monomer of UDA. Values of binding enthalpy (H) increase almost by a factor of 7 in going from chitobiose to chitotriose indicating the existence of three subsites in the combining site of UDA. The binding constant for chitotetrose and chitopentose increase without any further enhancement in the values of H indicating that for oligomers larger than chitotriose interaction is favoured entropically.     

Thermodynamic parameters of the interaction of Urtica dioica agglutinin with N-acetylglucosamine and its oligomers

The interaction between Urtica dioica agglutinin (UDA) and N-acetylglucosamine (GlcNAc) and its (1-4)-linked oligomers was studied by fluorescence titration and isothermal titration microcalorimetry. UDA possesses one significant binding site that can be measured calorimetrically. This site is composed of three subsites, each subsite accommodating one GlcNAc residue. The interaction is enthalpically driven, and the binding area of UDA is characterized by a H of interaction for a given oligosaccharide considerably smaller than that of wheat germ agglutinin (WGA), despite the fact that they both belong to a family of proteins composed entirely of hevein domains. Relatively high Cp values of the UDA-carbohydrate interactions and more favorable entropy term compared to WGA suggest that binding of the carbohydrate ligands by UDA has a higher hydrophobic component than that of WGA.     

Interactive binding to the two principal ligand binding sites of human serum albumin: effect of the neutral-to-base transition.

The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-L-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of lambda(max) and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a 'competitive' like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly 'independent'. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 A during the N-B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N-B transition.     

The specificity and function of the metal-binding sites in the integrin beta3 A-domain

The A-domains within integrin beta subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the beta3 A-domain, beta3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the beta3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to alphavbeta3 but not alphaIIbbeta3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the beta3 A-domain.     

Implication of tryptophan and tyrosine in the binding of p-nitrophenyl-alpha-D-mannopyranoside by concanavalin A. A radiolytic study.

Equilibrium dialysis has been used to study the effects of 60Co gamma-radiolysis on the carbohydrate binding site of Con A. Reaction of eaq- and OH. with Con A is accompanied by a decrease in the number of binding sites. Modification of the binding site by (Br)2- is independent of the initial concentration of Br-. Reaction of (SCN)2- with the protein, however, is dependent on the initial SCN- concentration. The results imply that tryptophan and possibly tyrosine are probably involved in the carbohydrate binding process.     

Physiochemical studies on interactions between DNA and RNA polymerase. Ultraviolet absorption measurements.

The interaction between Escherichia coli RNA polymerase and a restriction fragment of coliphage T7 DNA containing four promoter sites for the coli enzyme has been studied by difference uv absorption spectroscopy in a low ionic strength buffer containing 10 mm MgCl2 and 50 mM KCl. The binding of the enzyme to the DNA is accompanied by a hyperchromic shift which shows a maximum around 260 nm, and increases with increasing temperature in the temperature range studied (4-40 degrees C). Measurements were also carried out with whole T7 DNA and a restriction fragment containing no promoter site. A comparison of the results obtained with the various DNAs suggests that the binding of an RNA polymerase to a promoter site in the low ionic strength medium causes the disruption of a short segment of the DNA helix, of the order of ten pairs; the binding of an enzyme molecule to a promotor site appears to have a cooperative effect on the binding of the enzyme molecules to adjacent non-promoter sites with concomitant disruption of DNA base pairs.     

Binding of Lactose and Galactose to Native and Iodinated Ricin D

The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the low-affinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.

By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.

From these results, it was inferred that the binding site abolished by the iodination is the LA-binding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.     

Solution structure of a cyanovirin-N:Man alpha 1-2Man alpha complex: structural basis for high-affinity carbohydrate-mediated binding to gp120

BACKGROUND: Cyanovirin-N (CVN) is a novel, 11 kDa cyanobacterial protein that potently inhibits viral entry by diverse strains of HIV through high-affinity carbohydrate-mediated interactions with the viral envelope glycoprotein gp120. CVN contains two symmetry-related carbohydrate binding sites of differing affinities that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinities, the carbohydrates that also mediate CVN:gp120 binding. High-resolution structural studies of CVN in complex with a representative oligosaccharide are desirable for understanding the structural basis for this unprecedented specificity. RESULTS: We have determined by multidimensional heteronuclear NMR spectroscopy the three-dimensional solution structure of CVN in complex with two equivalents of the disaccharide Manalpha1-2Manalpha, a high-affinity ligand which represents the terminal-accessible disaccharide present in Man(8) D1D3 and Man(9). The structure reveals that the bound disaccharide adopts the stacked conformation, thereby explaining the selectivity for Man(8) D1D3 and Man(9) over other oligomannose structures, and presents two novel carbohydrate binding sites that account for the differing affinities of the two sites. The high-affinity site comprises a deep pocket that nearly envelops the disaccharide, while the lower-affinity site comprises a semicircular cleft that partially surrounds the disaccharide. The approximately 40 A spacing of the two binding sites provides a simple model for CVN:gp120 binding. CONCLUSIONS: The CVN:Manalpha1-2Manalpha complex provides the first high-resolution structure of a mannose-specific protein-carbohydrate complex with nanomolar affinity and presents a new carbohydrate binding motif, as well as a new class of carbohydrate binding protein, that facilitates divalent binding via a monomeric protein.     

The N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae binds specifically to mannose carbohydrates

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to D-mannose, α-methyl-D-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.     

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.     

Equilibrium and kinetic studies of the binding of tri- and tetra-anionic ligands to bovine serum albumin.

The binding of tri- and tetra-anionic azo dyes (Amaranth, Ponceau 4R, and Ponceau 6R) to bovine serum albumin (BSA) at pH = 7.0 and 25 degrees C has been studied by equilibrium dialysis, spectrophotometry, and by stopped-flow and temperature-jump methods. Equilibrium dialysis revealed that BSA has one primary binding site and about two secondary sites for each dye. The values of the binding constant for the primary site show that the stability of the complex at the primary site progressively increases with an increase in the number and the density of anionic charges on ligand. Kinetic data have been found to be consistent with a scheme in which a rapid bimolecular binding is followed by two isomerizations of the complex (in the case of Amaranth) or by one isomerization (in the cases of Ponceau 4R and Ponceau 6R). Equilibrium and rate constants for each step of the scheme were determined. From the results it was found that the increment of the number and the density of anionic charges on ligand accelerates the forward process of the final isomerization step but retards the backward one of it, resulting in the enhancement of the stability of the complex at the primary site. On the basis of these results and the structure of the ligands, the detailed binding mechanism has been discussed in the light of the electrostatic interaction between the ligands and the binding site on BSA.     

Ligand Binding Site Similarity Identification Based on Chemical and Geometric Similarity

The similarity comparison of binding sites based on amino acid between different proteins can facilitate protein function identification. However, Binding site usually consists of several crucial amino acids which are frequently dispersed among different regions of a protein and consequently make the comparison of binding sites difficult. In this study, we introduce a new method, named as chemical and geometric similarity of binding site (CGS-BSite), to compute the ligand binding site similarity based on discrete amino acids with maximum-weight bipartite matching algorithm. The principle of computing the similarity is to find a Euclidean Transformation which makes the similar amino acids approximate to each other in a geometry space, and vice versa. CGS-BSite permits site and ligand flexibilities, provides a stable prediction performance on the flexible ligand binding sites. Binding site prediction on three test datasets with CGS-BSite method has similar performance to Patch-Surfer method but outperforms other five tested methods, reaching to 0.80, 0.71 and 0.85 based on the area under the receiver operating characteristic curve, respectively. It performs a marginally better than Patch-Surfer on the binding sites with small volume and higher hydrophobicity, and presents good robustness to the variance of the volume and hydrophobicity of ligand binding sites. Overall, our method provides an alternative approach to compute the ligand binding site similarity and predict potential special ligand binding sites from the existing ligand targets based on the target similarity.     

Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin

A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.     

Discovery of ligands for Nurr1 by combined use of NMR screening with different isotopic and spin-labeling strategies

A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR (19)F signal perturbation was used to screen a small compound library and identify candidate ligands to the target of interest. Ligand dissociation constants were measured using a pegylated form of the protein, which resulted in a 2-fold increase in the strength of the saturation transfer difference signal. The initial small-molecule hits were further optimized by combining a residue-specific labeling strategy, to identify the specific sites of interaction with the protein, with a second site screening approach based on relaxation enhancement using a paramagnetic probe. The advantages of this combination strategy in the identification and optimization of weak binding chemical entities early in a program are illustrated with the discovery of a low micromolar ligand (K(d) = 20 microM) for Nurr1 and identification of the binding site location through residue-specific (15)N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-(13)C.     

ADP binding to TF1 and its subunits induces ultraviolet spectral changes

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.     

Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

Background

Nuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, N Heteronuclear Single Quantum Correlation (HSQC) experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately.

Methodology/Principal Findings

We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues. Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant () calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.

Conclusion/Significance

Detail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called “Auto-FACE”, which also allowed quantitative information of each interaction site and elucidation of binding mechanism.     

Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity

Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.     

Binding of Saccharides to Abrin-b Isolated from Abrus precatorius Seeds

The nature of the binding of saccharides to arbin-b, a toxic lectin isolated from Abrus precatorius seeds, was studied by equilibrium dialysis and fluorescence spectroscopy. Equilibrium dialysis data indicate that abrin-b has two saccharide-binding sites, a high affinity site (HA-site) and a low affinity site (LA-site), to which both galactopyranosides and N-acetylgalactosamine can bind. With excitation at 290 nm, abrin-b displayed a fluorescence spectrum with an emission maximum at 345 nm. Upon binding with specific saccharides, this spectrum shifted to a wavelength shorter by 5 nm, suggesting that saccharides bind to abrin-b in such a manner as to induce a change in the environment of the tryptophan residue or residues at or near the respective binding sites. From the variation of fluorescence at 320 nm with saccharide concentrations, the association constants for binding of saccharides to the respective sites were measured. The results suggest that the HA-site has a subsite favorable for saccharides having β-1,4 linked galactopyranoside at the non-reducing end like lactose in addition to the galactose-recognition site, while the LA-site may not have such a subsite.     

The relevance of N-linked glycosylation to the binding of a ligand to guanylate cyclase C.

The role of carbohydrate moieties at the N-linked glycosylation sites of guanylate cyclase C (GC-C), a receptor protein for guanylin, uroguanylin and heat-stable enterotoxin, in ligand binding and structural stability was examined using site-directed mutagenesis of the putative N-linked glycosylation sites in the extracellular domain (ECD) of porcine GC-C. For this purpose, eight mutant proteins of ECD (N9A, N20A, N56A, N172A, N261A, N284A, N334A and N379A) and six mutant proteins of the complete GC-C (N9A, S11A, N172A, T174A, N379A and T381A) were prepared, in which Ala replaced Asn, Ser and Thr at the N-linked glycosylation consensus sites. All the mutant proteins showed a ligand-binding affinity (K(d)) similar to those of the wild-type proteins, although the deletion of a carbohydrate moiety at each of the N-linked glycosylation sites affected the ligand-binding ability of ECD or GC-C to some degree. However, the mutant proteins of ECD (N379A) and GC-C (N379A and T381A) showed considerably decreased binding ability in the context of maximum capacity (B(max)) to a ligand, despite the fact that the expression levels of these mutant proteins were nearly the same as the wild-type proteins. Moreover, the mutant protein of ECD (N379A) was considerably less stable to a denaturant. These results clearly indicate a crucial role for the carbohydrate moiety at N379, which is located near the transmembrane region, in structural stability, the ability to bind to a ligand and the cyclase catalytic activity of GC-C, and provide a route for the elucidation of the mechanism of the interaction between GC-C and a ligand.