Comparative analysis of structural properties of the C-type-lectin-like domain (CTLD)

The superfamily of proteins containing the C-type-lectin-like domain (CTLD) is a group of abundant extracellular metazoan proteins characterized by evolutionary flexibility and functional versatility. Several CTLDs are also found in parasitic prokaryotes and viruses. The 37 distinct currently available CTLD structures demonstrate significant structural conservation despite low or undetectable sequence similarity. Our aim in this study was to perform an extensive comparative analysis of all available CTLD structures to establish the most conserved structural features of the fold, and to test and extend the early analysis of Drickamer. By implication, these features should be those critical for maintenance of integrity of the fold. By analyzing CTLD structures superimposed by several methods, we have established groups of conserved structural positions involved in fold maintenance but not in ligand binding; these are consistent with the fold's known functional flexibility. In addition to the well-recognized disulfide bridges, groups of conserved residues are involved in hydrophobic interactions stabilizing the core of the fold and the long loop region, and in an alpha2-beta1-beta5 polar interaction. Evaluation of the conclusions of the structure comparison study compared with alignments of all available human, mouse and Caenorhabditis elegans CTLD sequences showed that conservation patterns are preserved throughout the whole CTLD sequence space. Our observations provide an improved understanding of CTLD structure, and will help in identification of new CTLDs and the mechanisms that drive and constrain the coevolution of the structure and function of the fold.     

Characterization of sugar binding by the mannose receptor family member,Endo180

Members of the mannose receptor family, the mannose receptor, the phospholipase A(2) receptor, DEC-205, and Endo180, contain multiple C-type lectin-like domains (CTLDs) within a single polypeptide. In addition, at their N termini, all four family members contain a cysteine-rich domain similar to the R-type carbohydrate recognition domains of ricin. However, despite the common presence of multiple lectin-like domains, these four endocytic receptors have divergent ligand binding activities, and it is clear that the majority of these domains do not bind sugars. Here the functions of the lectin-like domains of the most recently discovered family member, Endo180, have been investigated. Endo180 is shown to bind in a Ca(2+)-dependent manner to mannose, fucose, and N-acetylglucosamine but not to galactose. This activity is mediated by one of the eight CTLDs, CTLD2. Competition assays indicate that the monosaccharide binding specificity of Endo180 CTLD2 is similar to that of mannose receptor CTLD4. However, additional experiments indicate that, unlike the cysteine-rich domain of the mannose receptor, the cysteine-rich domain of Endo180 does not bind sulfated sugars. Thus, although Endo180 and the mannose receptor are now both known to be mannose binding lectins, each receptor is likely to have a distinct set of glycoprotein ligands in vivo.     

Sweet Tooth, a novel receptor protein-tyrosine kinase with C-type lectin-like extracellular domains

A gene encoding a novel type of receptor protein-tyrosine kinase was identified in Hydra vulgaris. The extracellular portion of this receptor (which we have named Sweet Tooth) contains four C-type lectin-like domains (CTLDs). Comparison of the sequences of these domains with the sequences of the carbohydrate recognition domains of various vertebrate C-type lectins shows that Sweet Tooth CTLD1 and CTLD4 have amino acids in common with those shown to be involved in carbohydrate binding by the lectins. Comparison of sequences encoding CTLD1 from the Sweet Tooth genes from different species of Hydra shows variation in some of the conserved residues that participate in carbohydrate binding in C-type lectins. The Sweet Tooth gene is expressed widely in the Hydra polyp, and expression is particularly high in the endoderm of the tentacles. Treatment of polyps with peptides corresponding to sequences in the Sweet Tooth CTLDs results in the disintegration of the animal. These same peptides do not block adhesion or morphogenesis of Hydra cell aggregates.     

Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.     

The C-type lectin-like receptors of Dectin-1 cluster in natural killer gene complex

Natural killer gene complex (NKC) encodes a group of proteins with a single C-type lectin-like domain, (CTLD) which can be subdivided several subfamilies according to their structures and expression patterns. The receptors containing the conserved calcium binding sites in the CTLD fold belong to group II of C-type lectin superfamily and are expressed on myeloid cells and non- myeloid cells. The receptors lacking conserved calcium binding sites in the CTLD fold have evolved to bind ligands other than carbohydrates independently on calcium and thereby are named as C-type lectin-like receptors. The C-type lectin-like receptors are previously thought to be exclusively expressed on natural killer (NK) cells and enable NK cells to discriminate self, missing self or altered self. However, some C-type lectin-like receptors are identified in myeloid cells and are intensely investigated, recently. These myeloid C-type lectin-like receptors, especially Dectin-1 cluster, have a wide variety of ligands, including those of exogenous origin, and play important roles in the physiological functions and pathological processes including immune homeostasis, immune defenses, and immune surveillance. In this review, we summarize each member of the Dectin-1 cluster, including their structural profiles, expression patterns, signaling properties as well as known physiological functions.     

An extracellular calcium-binding domain in bacteria with a distant relationship to EF-hands

Extracellular Ca(2+)-dependent nuclease YokF from Bacillus subtilis and several other surface-exposed proteins from diverse bacteria are encoded in the genomes in two paralogous forms that differ by a approximately 45 amino acid fragment, which comprises a novel conserved domain. Sequence analysis of this domain revealed a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca(2+)-binding loop of the calmodulin-like EF-hand domains, suggesting an evolutionary relationship between them. Functions of many of the other proteins in which the novel domain, named Excalibur (extracellular calcium-binding region), is found, as well as a structural model of its conserved motif are consistent with the notion that the Excalibur domain binds calcium. This domain is but one more example of the diversity of structural contexts surrounding the EF-hand-like calcium-binding loop in bacteria. This loop is thus more widespread than hitherto recognized and the evolution of EF-hand-like domains is probably more complex than previously appreciated.     

Molecular architecture of mouse activating NKR-P1 receptors

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.     

Evolutionary analysis reveals collective properties and specificity in the C-type lectin and lectin-like domain superfamily

Members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily share a common fold and are involved in a variety of functions, such as generalized defense mechanisms against foreign agents, discrimination between healthy and pathogen-infected cells, and endocytosis and blood coagulation. In this work we used ConSurf, a computer program recently developed in our lab, to perform an evolutionary analysis of this superfamily in order to further identify characteristics of all or part of its members. Given a set of homologous proteins in the form of multiple sequence alignment (MSA) and an inferred phylogenetic tree, ConSurf calculates the conservation score in every alignment position, taking into account the relationships between the sequences and the physicochemical similarity between the amino acids. The scores are then color-coded onto the three-dimensional structure of one of the homologous proteins. We provide here and at http://ashtoret.tau.ac.il/ approximately sharon a detailed analysis of the conservation pattern obtained for the entire superfamily and for two subgroups of proteins: (a) 21 CTLs and (b) 11 heterodimeric CTLD toxins. We show that, in general, proteins of the superfamily have one face that is constructed mostly of conserved residues and another that is not, and we suggest that the former face is involved in binding to other proteins or domains. In the CTLs examined we detected a region of highly conserved residues, corresponding to the known calcium- and carbohydrate-binding site of the family, which is not conserved throughout the entire superfamily, and in the CTLD toxins we found a patch of highly conserved residues, corresponding to the known dimerization region of these proteins. Our analysis also detected patches of conserved residues with yet unknown function(s).     

Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor

The mannose receptor family comprises four members in mammals, Endo180 (CD280), DEC-205 (CD205), phospholipase A(2) receptor (PLA(2)R) and the mannose receptor (MR, CD206), whose extracellular portion contains a similar domain arrangement: an N-terminal cysteine-rich domain (CysR) followed by a single fibronectin type II domain (FNII) and 8-10 C-type lectin-like domains (CTLDs). These proteins mediate diverse functions ranging from extracellular matrix turnover through collagen uptake to homeostasis and immunity based on sugar recognition. Endo180 and the MR are multivalent transmembrane receptors capable of interacting with multiple ligands; in both receptors FNII recognizes collagens, and a single CTLD retains lectin activity (CTLD2 in Endo180 and CTLD4 in MR). It is expected that the overall conformation of these multivalent molecules would deeply influence their function as the availability of their binding sites could be altered under different conditions. However, conflicting reports have been published on the three-dimensional arrangement of these receptors. Here, we have used single particle electron microscopy to elucidate the three-dimensional organization of the MR and Endo180. Strikingly, we have found that both receptors display distinct three-dimensional structures, which are, however, conceptually very similar: a bent and compact conformation built upon interactions of the CysR domain and the lone functional CTLD. Biochemical and electron microscopy experiments indicate that, under a low pH mimicking the endosomal environment, both MR and Endo180 experience large conformational changes. We propose a structural model for the mannose receptor family where at least two conformations exist that may serve to regulate differences in ligand selectivity.     

C-Type lectin-like domains in Caenorhabditis elegans: predictions from the complete genome sequence

Protein modules related to the C-type carbohydrate-recognition domains of animal lectins are found in at least 125 proteins encoded in the Caenorhabditis elegans genome. Within these proteins, 183 C-type lectin-like domains (CTLDs) have been identified. The proteins have been classified based on the overall arrangement of modules within the polypeptides and based on sequence similarity between the CTLDs. The C.elegans proteins generally have different domain organization from known mammalian proteins containing CTLDs. Most of the CTLDs are divergent in sequence from those in mammalian proteins. However, 19 show conservation of most of the amino acid residues that ligate Ca(2+)to form a carbohydrate-binding site in vertebrate C-type carbohydrate-recognition domains. Seven of these domains are particularly similar in sequence to mannose- and N-acetylglucosamine-binding domains in the vicinity of this Ca(2+)site.     

Caulollins from Caulobacter crescentus, a pair of partially unstructured proteins of betagamma-crystallin superfamily, gain structure upon binding calcium

The betagamma-crystallin superfamily comprises members from various taxa and species, which have similar domain topologies as that of lens beta- and gamma-crystallins. We have studied new microbial members of this understudied betagamma-crystallin superfamily from the bacterium Caulobacter crescentus. These proteins, which we named "caulollins", are paralogues with a single betagamma-crystallin domain, made up of two Greek key motifs with AB-type arrangement seen in gamma-crystallin. The second Greek key motif has Cys in place of a generally conserved Phe/Tyr residue, and the Tyr corner, considered important for the proper betagamma-crystallin fold, is missing, making this a sequentially diverse atypical betagamma-crystallin domain. This atypical domain binds two Ca2+ with moderate affinity (0.8-20 microM). In apo form, caulollins are partially unstructured proteins and gain structure upon binding Ca2+. Unlike many other microbial betagamma-crystallin domains, this domain is monomeric, though in the presence of Ca2+ it becomes more compact. Ca2+ binding increases the intrinsic stability of proteins, suggesting the role of Ca2+ as an extrinsic stabilizer. N-Terminal extension does not play any role in modulating Ca2+ binding, intrinsic stability, or oligomerization. We noted that there are several such variant domains in the genomes of unrelated species. It appears that caulollins along with these members form a subfamily in the betagamma-crystallin superfamily that would be partially unstructured in apo form, unlike many other domains from lens or microbial crystallins. This work further suggests that Ca2+ binding is a widespread feature of the betagamma-crystallin superfamily.     

A targeted deletion in the endocytic receptor gene Endo180 results in a defect in collagen uptake

The four members of the mannose receptor family (the mannose receptor, the M-type phospholipase A₂ receptor, DEC-205 and Endo180) share a common extracellular arrangement of an amino-terminal cysteine-rich domain followed by a fibronectin type II (FNII) domain and multiple C-type lectin-like domains (CTLDs). In addition, all have a short cytoplasmic domain, which mediates their constitutive recycling between the plasma membrane and the endosomal apparatus, suggesting that these receptors function to internalize ligands for intracellular delivery. We have generated mice with a targeted deletion of Endo180 exons 2–6 and show that this mutation results in the efficient expression of a truncated Endo180 protein that lacks the cysteine-rich domain, the FNII domain and CTLD1. Analysis of embryonic fibroblasts reveals that this mutation does not disrupt the C-type lectin activity that is mediated by CTLD2, but results in cells that have a defect in collagen binding and internalization and an impaired migratory phenotype.     

Structural basis for recognition of high mannose type glycoproteins by mammalian transport lectin VIP36

VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca(2+) and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel beta-sandwich and binds one Ca(2+) adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca(2+) ion orients the side chains of Asp(131), Asn(166), and His(190) for carbohydrate binding. This result explains the Ca(2+)-dependent carbohydrate binding of this protein. The Man-alpha-1,2-Man-alpha-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca(2+)-dependent and D1 arm-specific manner.     

Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.     

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the μ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca(2+). The Ca(2+)-dependent interaction between the μ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca(2+) binding loop region that modulate the Ca(2+)-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca(2+)-binding loop region of C2B domain. The results indicate that Ca(2+)-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca(2+)-bound Synaptotagmin-1 associated synaptic vesicles.     

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.     

Role of the hexapeptide disulfide loop in the gamma-carboxyglutamic acid domain of protein C in Ca2+-mediated structural and functional properties

The anticoagulant and immunomodulatory effects of protein C (PC) rely on the presence of the N-terminal gamma-carboxyglutamic acid (Gla) domain. This domain is strongly conserved among vitamin K-dependent blood proteins and, in addition to a high relative content of Gla, contains a hexapeptide disulfide loop between Cys residues 17 and 22. In the present study, the contribution of the hexapeptide loop toward Gla domain structure and function was evaluated using wild-type and Cys17/Cys22-alkylated synthetic peptide analogues of the 47-residue Gla domain/helical stack of PC. Circular dichroism and intrinsic fluorescence measurements revealed significant differences in the metal ion-dependent conformations of the two peptides. Disruption of the disulfide loop slightly altered the capacity of the peptide to interact with acidic phospholipid (PL) vesicles. The affinity of the alkylated peptide for soluble endothelial protein C receptor (EPCR), as demonstrated by surface plasmon resonance studies, was increased compared with the wild-type species, although total binding was compromised. These results suggest that the disulfide loop of PC contributes to the overall Ca(2+)-dependent conformation but is not strictly required for PL membrane binding or EPCR recognition.     

Structure and properties of the Ca(2+)-binding CUB domain, a widespread ligand-recognition unit involved in major biological functions

CUB domains are 110-residue protein motifs exhibiting a β-sandwich fold and mediating protein-protein interactions in various extracellular proteins. Recent X-ray structural and mutagenesis studies have led to the identification of a particular CUB domain subset, cbCUB (Ca(2+)-binding CUB domain). Unlike other CUB domains, these harbour a homologous Ca(2+)-binding site that underlies a conserved binding site mediating ionic interaction between two of the three conserved acidic Ca(2+) ligands and a basic (lysine or arginine) residue of a protein ligand, similar to the interactions mediated by the low-density lipoprotein receptor family. cbCUB-mediated protein-ligand interactions usually involve multipoint attachment through several cbCUBs, resulting in high-affinity binding through avidity, despite the low affinity of individual interactions. The aim of the present review is to summarize our current knowledge about the structure and functions of cbCUBs, which represent the majority of the known CUB repertoire and are involved in a variety of major biological functions, including immunity and development, as well as in various cancer types. Examples discussed in the present review include a wide range of soluble and membrane-associated human proteins, as well as some archaeal and invertebrate proteins. The fact that these otherwise unrelated proteins share a common Ca(2+)-dependent ligand-binding ability suggests a mechanism inherited from very primitive ancestors. The information provided in the present review should stimulate further investigations on the crucial interactions mediated by cbCUB-containing proteins.     

Refolding and characterization of the functional ligand-binding domain of human lectin-like oxidized LDL receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a type II membrane protein that can recognize a variety of structurally unrelated macromolecules, plays an important role in host defense and is implicated in atherogenesis. To understand the interaction between human LOX-1 and its ligands, in this study the functional C-type lectin-like domain (CTLD) of LOX-1 was reconstituted at high efficiency from inactive aggregates in Escherichia coli using a refolding technique based on an artificial chaperone. The CD spectra of the purified domain suggested that the domain has alpha-helical structure and the blue shift of Trp residues was observed on refolding of the domain. Like wild-type hLOX-1, the refolded CTLD domain was able to bind modified LDL. Thus, even though CTLD contains six Cys residues that form disulfide bonds, it recovered its specific binding ability on refolding. This suggests that the correct disulfide bonds in CTLD were formed by the artificial chaperone technique. Although the domain lacked N-glycosylation, it showed high affinity for its ligand in surface plasmon resonance experiments. Thus, unglycosylated CTLD is sufficient for binding modified LDL.