Expression of glucagon receptors in tetracycline-inducible HEK293S GnT1- stable cell lines: an approach toward purification of receptor protein for structural studies

Glucagon is a 29-amino acid polypeptide hormone secreted by pancreatic A cells. Together with insulin, it is an important regulator of glucose metabolism. Type 2 diabetes is characterized by reduced insulin secretion from pancreatic B cells and increased glucose output by the liver which has been attributed to abnormally elevated levels of glucagon. The glucagon receptor (GR) is a member of family B G protein-coupled receptors, ligands for which are peptides composed of 30-40 amino acids. The impetus for studying how glucagon interacts with its membrane receptor is to gain insight into the mechanism of glucagon action in normal physiology as well as in diabetes mellitus. The principal approach toward this goal is to design and synthesize antagonists of glucagon that will bind with high affinity to the GR but will not activate it. Site-directed mutagenesis of the GR has provided some insight into the interactions between glucagon and GR. The rational design of potent antagonists has been hampered by the lack of structural information on receptor-bound glucagon. To obtain adequate amounts of receptor protein for structural studies, a tetracycline-inducible HEK293S GnT1(-) cell line that stably expresses human GR at high-levels was developed. The recombinant receptor protein was characterized, solubilized, and isolated by one-step affinity chromatography. This report describes a feasible approach for the preparation of human GR and other family B GPCRs in the quantities required for structural studies.     

Class-B GPCR activation: is ligand helix-capping the key?

The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro- and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCR-ligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.     

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with (beta)arr2. However, when the wild-type receptor was coexpressed with both (beta)arr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level.     

Sequence,Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

Elucidation of the role of peptide linker in calcium-sensing receptor activation process

Family 3 G-protein-coupled receptors (GPCRs), which includes metabotropic glutamate receptors (mGluRs), sweet and "umami" taste receptors (T1Rs), and the extracellular calcium-sensing receptor (CaR), represent a distinct group among the superfamily of GPCRs characterized by large amino-terminal extracellular ligand-binding domains (ECD) with homology to bacterial periplasmic amino acid-binding proteins that are responsible for signal detection and receptor activation through as yet unresolved mechanism(s) via the seven-transmembrane helical domain (7TMD) common to all GPCRs. To address the mechanism(s) by which ligand-induced conformational changes are conveyed from the ECD to the 7TMD for G-protein activation, we altered the length and composition of a 14-amino acid linker segment common to all family 3 GPCRs except GABA(B) receptor, in the CaR by insertion, deletion, and site-directed mutagenesis of specific highly conserved residues. Small alterations in the length and composition of the linker impaired cell surface expression and abrogated signaling of the chimeric receptors. The exchange of nine amino acids within the linker of CaR with the homologous sequence of mGluR1, however, preserved receptor function. Ala substitution for the four highly conserved residues within this amino acid sequence identified a Leu at position 606 of the CaR critical for cell surface expression and signaling. Substitution of Leu(606) for Ala resulted in impaired cell surface expression. However, Ile and Val substitutions displayed strong activating phenotypes. Disruption of the linker by insertion of nine amino acids of a random-coiled structure uncoupled the ECD from regulating the 7TMD. These data are consistent with a model of receptor activation in which the peptide linker, and particularly Leu(606), provides a critical interaction for the CaR signal transmission, a finding likely to be relevant for all family 3 GPCRs containing this conserved motif.     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.     

Molecular basis of secretin docking to its intact receptor using multiple photolabile probes distributed throughout the pharmacophore

The molecular basis of ligand binding and activation of family B G protein-coupled receptors is not yet clear due to the lack of insight into the structure of intact receptors. Although NMR and crystal structures of amino-terminal domains of several family members support consistency in general structural motifs that include a peptide-binding cleft, there are variations in the details of docking of the carboxyl terminus of peptide ligands within this cleft, and there is no information about siting of the amino terminus of these peptides. There are also no empirical data to orient the receptor amino terminus relative to the core helical bundle domain. Here, we prepared a series of five new probes, incorporating photolabile moieties into positions 2, 15, 20, 24, and 25 of full agonist secretin analogues. Each bound specifically to the receptor and covalently labeled single distinct receptor residues. Peptide mapping of labeled wild-type and mutant receptors identified that the position 15, 20, and 25 probes labeled residues within the distal amino terminus of the receptor, whereas the position 24 probe labeled the amino terminus adjacent to TM1. Of note, the position 2 probe labeled a residue within the first extracellular loop of the receptor, a region not previously labeled, providing an important new constraint for docking the amino-terminal region of secretin to its receptor core. These additional experimentally derived constraints help to refine our understanding of the structure of the secretin-intact receptor complex and provide new insights into understanding the molecular mechanism for activation of family B G protein-coupled receptors.     

Homologous regulation of melanocortin-1 receptor (MC1R) expression in melanoma tumor cells in vivo

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist alpha-melanocyte-stimulating hormone (alpha-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B 16F1 cells, a single injection of 50 to 500 microg alpha-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16-24 h. By continuous administration of alpha-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.     

High yield production of recombinant native and modified peptides exemplified by ligands for G-protein coupled receptors

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins and attract pharmaceutical interest as therapeutic targets. Two examples of class B GPCRs that are involved in metabolic diseases are the Parathyroid hormone receptor 1 (PTHR1) and the Glucagon-like-peptide-1 receptor (GLP-1R) which play central roles in osteoporosis and diabetes mellitus type II, respectively. Class B GPCRs are characterised by a large extracellular N-terminal domain with a typical disulfide bridge pattern. This domain is responsible for the binding of peptide hormone ligands. Here we report the recombinant expression of these ligands in natural and several modified forms for their use in functional assays, NMR analyses or affinity purification of receptor/ligand complexes for crystallisation. Applying the SUMO system, low cost expression of soluble fusion-proteins is achieved. Moreover, via the SUMO cleavage site, the authentic N-terminal sequence which is essential for ligand-receptor interactions can be obtained. Purification of the peptide by RP-HPLC results in >98% pure preparations. The strategy can also be adopted for many other purposes, especially if small peptides are needed at either large amounts or with specific features like isotope, affinity or fluorescent labels. Furthermore, for the growing demand for therapeutic peptides, this method could represent a straightforward production process.     

Paralog-divergent Features May Help Reduce Off-target Effects of Drugs: Hints from Glucagon Subfamily Analysis

Side effects from targeted drugs remain a serious concern. One reason is the nonselective binding of a drug to unintended proteins such as its paralogs, which are highly homologous in sequences and have similar structures and drug-binding pockets. To identify targetable differences between paralogs, we analyzed two types(type-I and type-II) of functional divergence between two paralogs in the known target protein receptor family G-protein coupled receptors(GPCRs) at the amino acid level. Paralogous protein receptors in glucagon-like subfamily, glucagon receptor(GCGR) and glucagon-like peptide-1 receptor(GLP-1R), exhibit divergence in ligands and are clinically validated drug targets for type 2 diabetes. Our data showed that type-II amino acids were significantly enriched in the binding sites of antagonist MK-0893 to GCGR, which had a radical shift in physicochemical properties between GCGR and GLP-1R. We also examined the role of type-I amino acids between GCGR and GLP-1R. The divergent features between GCGR and GLP-1R paralogs may be helpful in their discrimination, thus enabling the identification of binding sites to reduce undesirable side effects and increase the target specificity of drugs.     

Ligand-specific changes in M3 muscarinic acetylcholine receptor structure detected by a disulfide scanning strategy

G protein-coupled receptor (GPCR) function can be modulated by different classes of ligands including full and inverse agonists. At present, little is known about the conformational changes that agonist ligands induce in their target GPCRs. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced structural changes in a series of cysteine (Cys)-substituted mutant M 3 muscarinic acetylcholine receptors. One of our goals was to study whether the cytoplasmic end of transmembrane domain V (TM V), a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change, similar to the adjacent region of TM VI. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated 20 double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492). These receptors were transiently expressed in COS-7 cells and subsequently characterized in pharmacological and disulfide cross-linking studies. Our cross-linking data, in conjunction with a three-dimensional model of the M 3 muscarinic receptor, indicate that M 3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors. The present study provides novel information about the dynamic changes that accompany M 3 receptor activation and how the receptor conformations induced (or stabilized) by full versus inverse muscarinic agonists differ from each other at the molecular level. Because all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from the present study should be of broad general relevance.     

HOMOLOGOUS REGULATION OF MELANOCORTIN-1 RECEPTOR (MC1R) EXPRESSION IN MELANOMA TUMOR CELLS IN VIVO

ABSTRACT

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist α-melanocyte-stimulating hormone (α-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B16F1 cells, a single injection of 50 to 500?µg α-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16–24?h. By continuous administration of α-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.     

Bioinformatics and type II G-protein-coupled receptors

The best known family B, or Type II, G-protein-coupled receptors (GPCRs) recognize peptides as ligands. The receptors for corticotrophin-releasing factor, parathyroid hormone and secretin typify this group. However, there are only 15 such GPCRs. Many other receptors share sequence homology and have been assigned to this family. The ten 'Frizzled' and one 'Smoothened' receptors show the lowest sequence homology and are not necessarily G-protein coupled. Drosophila genetics have enabled our understanding of their biology. In contrast, relatively little is known about the largest group with family B, the 33 'large amino termini' or large N-terminal family B seven-transmembrane (LNB 7TM) receptors. This review highlights the similarities found between family B receptors and provides a classification of LNB 7TM receptors.     

Glucagon-induced desensitization of adenylyl cyclase in primary cultures of chick hepatocytes. Evidence for multiple pathways

Chick hepatocytes in primary culture have been used to study the homologous and heterologous pathways of glucagon-induced desensitization of adenylyl cyclase. Scatchard analysis and guanine nucleotide effects on dissociation kinetics indicate that the initial phase of homologous desensitization, an increase in low affinity glucagon receptors due to the rapid uncoupling of the receptor from Gs, is essentially complete within 5 min. These receptors recouple within 20 min upon removal of glucagon. Upon prolonged (2 h or more) exposure of hepatocytes to glucagon, disappearance of low affinity receptors from cell surface membranes constitutes the second phase of homologous desensitization. Recovery of these lost and presumably internalized receptors requires more than 12 h following the removal of glucagon but is not dependent on new protein synthesis. The heterologous phase of desensitization is slower, requiring 20-30 min of glucagon treatment to reach completion. Stimulation of adenylyl cyclase by hormonal and nonhormonal effectors is similarly reduced, indicating a common defect in this desensitized state. Agonist occupancy of other hormone receptors coupled to adenylyl cyclase in hepatocytes, such as beta-adrenergic, prostaglandin E1, and vasoactive intestinal peptide, results in heterologous desensitization. Heterologous desensitization is rapidly reversed (within 30 min) upon partial removal of glucagon, under conditions allowing the maintenance of the homologously desensitized state. Neither onset of nor recovery from heterologous desensitization requires protein synthesis. These data indicate that homologous and heterologous desensitization occurs by independent mechanisms. Homologous desensitization involves uncoupling of the glucagon receptor from Gs, followed by removal of these uncoupled receptors from the cell surface. Heterologous desensitization represents a second level of cellular control of hormonal responsiveness to be turned on when the cell is subjected to prolonged hormonal stimulation and withdrawn when hormone levels are lowered.     

Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.     

Site of action of a pentapeptide agonist at the glucagon-like peptide-1 receptor. Insight into a small molecule agonist-binding pocket

The development of small molecule agonists for class B G protein-coupled receptors (GPCRs) has been quite challenging. With proof-of-concept that exenatide, the parenterally administered peptide agonist of the glucagon-like peptide-1 (GLP1) receptor, is an effective treatment for patients with diabetes mellitus, the development of small molecule agonists could have substantial advantages. We previously reported a lead for small molecule GLP1 receptor agonist development representing the pentapeptide NRTFD. In this work, we have prepared an NRTFD derivative incorporating a photolabile benzoylphenylalanine and used it to define its site of action. This peptide probe was a full agonist with potency similar to NRTFD, which bound specifically and saturably to a single, distinct site within the GLP1 receptor. Peptide mapping using cyanogen bromide and endoproteinase Lys-C cleavage of labeled wild type and M397L mutant receptor constructs identified the site of covalent attachment of NRTFD within the third extracellular loop above the sixth transmembrane segment (TM6). This region is the same as that identified using an analogous photolabile probe based on secretin receptor sequences, and has been shown in mutagenesis studies to be important for natural agonist action of several members of this family. While these observations suggest that small molecule ligands can act at a site bordering the third extracellular loop to activate this class B GPCR, the relationship of this site to the site of action of the amino-terminal end of the natural agonist peptide is unclear.     

Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

G protein-coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins. Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains. Downregulation of mth increases the life span of Drosophila melanogaster; inhibitors of Mth signaling should therefore enhance longevity. We used mRNA display selection to identify high-affinity (K(d) = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane domains. Flies constitutively expressing a Mth antagonist peptide have a robust life span extension, which suggests that the peptides inhibit Mth signaling in vivo. Our work thus provides new life span-extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.     

NCD3G: a novel nine-cysteine domain in family 3 GPCRs

The NCD3G [for nine-cysteine domain of family 3 G-protein-coupled receptors (GPCRs)] domain is a novel protein domain that is conserved in family 3 GPCRs, including metabotropic glutamate receptors, calcium-sensing receptors, pheromone receptors and taste receptors, with the exception of GABA(B) receptors. The NCD3G domain contains nine highly conserved cysteine residues. Structural predictions suggest that NCD3G might possess four beta strands and three disulfide bridges. The structural model of NCD3G highlights the conserved residues co-segregated with certain familial diseases.