The 1.5 Å crystal structure of human receptor for advanced glycation endproducts (RAGE) ectodomains reveals unique features determining ligand binding

Interaction of the pattern recognition receptor, RAGE with key ligands such as advanced glycation end products (AGE), S100 proteins, amyloid β, and HMGB1 has been linked to diabetic complications, inflammatory and neurodegenerative disorders, and cancer. To help answer the question of how a single receptor can recognize and respond to a diverse set of ligands we have investigated the structure and binding properties of the first two extracellular domains of human RAGE, which are implicated in various ligand binding and subsequent signaling events. The 1.5-Å crystal structure reveals an elongated molecule with a large basic patch and a large hydrophobic patch, both highly conserved. Isothermal titration calorimetry (ITC) and deletion experiments indicate S100B recognition by RAGE is an entropically driven process involving hydrophobic interaction that is dependent on Ca²⁺ and on residues in the C′D loop (residues 54–67) of domain 1. In contrast, competition experiments using gel shift assays suggest that RAGE interaction with AGE is driven by the recognition of negative charges on AGE-proteins. We also demonstrate that RAGE can bind to dsDNA and dsRNA. These findings reveal versatile structural features of RAGE that help explain its ability to recognize of multiple ligands.     

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca(2+)-loaded S100B was determined at 1.9 A resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.     

Interaction of S100A13 with C2 domain of receptor for advanced glycation end products (RAGE)

S100A13 is involved in several key biological functions like angiogenesis, tumor formation and cell apoptosis. It is a homodimeric protein that belongs to the S100 protein family. S100A13 is co-expressed with acidic fibroblast growth factor (FGF1) and interleukin-1α which are key angiogenesis inducers. The S100 proteins have been shown to be involved in several cellular functions such as calcium homeostasis, cell growth and differentiation dynamic of cytoskeleton. Its biological functions are mainly mediated through the receptor for advanced glycation end products (RAGE) signaling. RAGE is involved in inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling upon binding of different ligands, such as S100 proteins, glycated proteins, and HMGB1. RAGE signaling is complex, and it depends on the cell type and concentration of the ligand. Molecular level interactions of RAGE and S100 proteins are useful to understand the RAGE signaling diversity. In this report we focus on the molecular level interactions of S100A13 and RAGE C2 domain. The binding between RAGE C2 and S100A13 is moderately strong (K_d ~ 1.3 μM). We have solved the solution structure of the S100A13–RAGE C2 complex and pronounce the interface regions in S100A13–RAGE C2 complex which are helpful for drug development of RAGE induced diseases.     

The extracellular region of the receptor for advanced glycation end products is composed of two independent structural units

The receptor for advanced glycation end products (RAGE) is an important cell surface receptor being pursued as a therapeutic target because it has been implicated in complications arising from diabetes and chronic inflammatory conditions. RAGE is a single membrane spanning receptor containing a very small approximately 40 residue cytosolic domain and a large extracellular region composed of 3 Ig-like domains. In this study, high level bacterial expression systems and purification protocols were generated for the extracellular region of RAGE (sRAGE) and the five permutations of single and tandem domain constructs to enable biophysical and structural characterization of its tertiary and quaternary structure. The structure and stability of each of these six protein constructs was assayed by biochemical methods including limited proteolysis, dynamic light scattering, CD, and NMR. A homology model of sRAGE was constructed to aid in the interpretation of the experimental data. Our results show that the V and C1 domains are not independent domains, but rather form an integrated structural unit. In contrast, C2 is attached to VC1 by a flexible linker and is fully independent. The interaction with a known RAGE ligand, Ca2+-S100B, was mapped to VC1, with the major contribution from the V domain but clearly defined secondary effects from the C1 domain. The implications of these results are discussed with respect to models for RAGE signaling.     

S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains

S100 proteins are EF-hand calcium-binding proteins with various intracellular functions including cell proliferation, differentiation, migration, and apoptosis. Some S100 proteins are also secreted and exert extracellular paracrine and autocrine functions. Experimental results suggest that the receptor for advanced glycation end products (RAGE) plays important roles in mediating S100 protein-induced cellular signaling. Here we compared the interaction of two S100 proteins, S100B and S100A6, with RAGE by in vitro assay and in culture of human SH-SY5Y neuroblastoma cells. Our in vitro binding data showed that S100B and S100A6, although structurally very similar, interact with different RAGE extracellular domains. Our cell assay data demonstrated that S100B and S100A6 differentially modulate cell survival. At micromolar concentration, S100B increased cellular proliferation, whereas at the same concentration, S100A6 triggered apoptosis. Although both S100 proteins induced the formation of reactive oxygen species, S100B recruited phosphatidylinositol 3-kinase/AKT and NF-kappaB, whereas S100A6 activated JNK. More importantly, we showed that S100B and S100A6 modulate cell survival in a RAGE-dependent manner; S100B specifically interacted with the RAGE V and C(1) domains and S100A6 specifically interacted with the C(1) and C(2) RAGE domains. Altogether these results highlight the complexity of S100/RAGE cellular signaling.     

Resonance assignments of Ca2+-bound human S100A11

The S100 family belongs to the EF-hand calcium-binding proteins regulating a wide range of important cellular processes via protein–protein interactions. Most S100 proteins adopt a conformation of non-covalent homodimer for their functions. Calcium binding to the EF-hand motifs of S100 proteins is essential for triggering the structural changes, promoting exposure of hydrophobic regions necessary for target protein interactions. S100A11 is a protein found in diverse tissues and possesses multiple functions upon binding to different target proteins. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. However, the three-dimensional structure of human S100A11 containing 105 amino acids is still not available for further interaction studies. To determine the solution structure, for the first time we report the ¹H, ¹⁵N and ¹³C resonance assignments and protein secondary structure prediction of human S100A11 dimer in complex with calcium using a variety of triple resonance NMR experiments and the chemical shift index (CSI) method, respectively.     

RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins

The S100 protein family comprises at least 25 members which, with the exception of S100G, act as Ca2+-sensor proteins that participate in Ca2+ signal transduction by interacting with target proteins thereby modifying their activities. S100 proteins are expressed in vertebrates exclusively, display a cell-specific distribution, and regulate a large variety of intracellular activities. Some S100 proteins are released by a non-classical pathway and exert regulatory effects on several cell types. The receptor for advanced glycation end products (RAGE) has been shown to transduce extracellular effects of S100B, S100A4, S100A6, S100A11, S100A12, S100A13 and S100P. However, some S100 proteins can signal by engaging RAGE as well as non-RAGE receptors. Immune cells (i.e., monocytes/macrophages/microglia, neutrophils and lymphocytes), activated endothelial and vascular smooth muscle cells, neurons, astrocytes, chondrocytes and pancreatic tumor cells are the cell types reported to respond to certain S100 proteins via RAGE engagement. In general, relatively high concentrations of S100 proteins are required for activation of RAGE in responsive cells. S100B is unique in that it can engage RAGE in neurons at low and high concentrations with trophic and toxic effects, respectively, and S100A4 stimulates matrix metalloproteinase 13 release from chondrocytes at nanomolar doses in a RAGE-mediated manner. Oligomerization of S100 proteins under the non-reducing, high-Ca2+ conditions found extracellularly appears to play a relevant role in RAGE activation, and binding of at least S100A12 and S100B results in RAGE oligomerization. Thus, S100/RAGE interactions might have important consequences during development and in tissue homeostasis as well as in inflammatory, degenerative and tumor processes.     

Hexameric calgranulin C (S100A12) binds to the receptor for advanced glycated end products (RAGE) using symmetric hydrophobic target-binding patches

Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is the receptor for advanced glycation products (RAGE). The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. Here, by using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface. Ca2+ binding creates two symmetric hydrophobic surfaces on Ca2+-calgranulin C that allow calgranulin C to bind to the C-type immunoglobulin domain of RAGE. Apo-calgranulin C also binds to sRAGE using a completely different surface and with substantially lower affinity, thus underscoring the role of Ca2+ binding to S100 proteins as a molecular switch. By using native gel electrophoresis, chromatography, and fluorescence spectroscopy, we established that sRAGE forms tetramers that bind to hexamers of Ca2+-calgranulin C. This arrangement creates a large platform for effectively transmitting RAGE-dependent signals from extracellular S100 proteins to the cytoplasmic signaling complexes.     

Carboxylated N‐glycans on RAGE promote S100A12 binding and signaling

The receptor for advanced glycation end products (RAGE) is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V‐domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than 10‐fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan‐enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non‐glycosylated sRAGE expressed in E. coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12‐mediated NF‐κB signaling in HeLa cells expressing full‐length RAGE. These results demonstrate that carboxylated N‐glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding. J. Cell. Biochem. 110: 645–659, 2010. © 2010 Wiley‐Liss, Inc.     

Interaction between S100P and the anti-allergy drug cromolyn

The S100P protein has been known to mediate cell proliferation by binding the receptor for advanced glycation end products (RAGE) to activate signaling pathways, such as the extracellular regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. S100P/RAGE signaling is involved in a variety of diseases, such as cancer, metastasis, and diabetes. Cromolyn is an anti-allergy drug that binds S100P to block the interaction between S100P and RAGE. In the present study, we characterized the properties of the binding between cromolyn and calcium-bound S100P using various biophysical techniques. The binding affinity for S100P and cromolyn was measured to be in the millimolar range by fluorescence spectroscopy. NMR-HSQC titration experiments and HADDOCK modeling was employed to determine the spatial structure of the proposed heterotetramer model of the S100P–cromolyn complex. Additional MD simulation results revealed the important properties in the complex stability and conformational flexibility of the S100P–cromolyn complex. This proposed model has provided an understanding of the molecular level interactions of S100P–cromolyn complex.     

RAGE recycles at the plasma membrane in S100B secretory vesicles and promotes Schwann cells morphological changes

RAGE is a multiligand receptor of the immunoglobulin superfamily involved in regeneration of injured peripheral nerve and cell motility. RAGE is implicated in the development of various chronic diseases, such as neurodegenerative disorders, inflammatory responses, and diabetic complications. The correlation between RAGE endocytic trafficking and RAGE function is still uninvestigated. S100B is one of the ligands of RAGE. The molecular mechanisms responsible of S100B translocation in exocytic vesicles are still poorly investigated. In the present study we elucidate the role of RAGE endocytic trafficking in promoting S100B secretion in Schwann cells. Here we show that RAGE-induced secretion of S100B requires phosphorylated caveolin1-dependent endocytosis of RAGE. Endocytosis of RAGE in response to ligand binding promotes the fusion of endosomes with S100B-positive secretory vesicles. Src promotes the fusion of endosomes with S100B-secretory vesicles. Inhibition of src induces RAGE degradation. RAGE-mediated src activation induces cav1 phosphorylation and relocalization in the perinuclear compartment. RAGE signaling and recycling are required for S100-induced Schwann cells morphological changes and are inhibited by high-glucose, suggesting a possible link between diabetes and peripheral nerve injury. Indeed, high glucose inhibits RAGE-mediated src activation. Src inhibition blocks RAGE recycling, S100B secretion, and morphological changes. In summary, we identified a novel pathway of vesicular trafficking required for the amplification of RAGE signaling and cytoskeleton dynamics that is potentially involved in the regeneration of injured peripheral nerve.     

Modeling the Interaction between Quinolinate and the Receptor for Advanced Glycation End Products (RAGE): Relevance for Early Neuropathological Processes

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor involved in neurodegenerative and inflammatory disorders. RAGE induces cellular signaling upon binding to a variety of ligands. Evidence suggests that RAGE up-regulation is involved in quinolinate (QUIN)-induced toxicity. We investigated the QUIN-induced toxic events associated with early noxious responses, which might be linked to signaling cascades leading to cell death. The extent of early cellular damage caused by this receptor in the rat striatum was characterized by image processing methods. To document the direct interaction between QUIN and RAGE, we determined the binding constant (K_b) of RAGE (VC1 domain) with QUIN through a fluorescence assay. We modeled possible binding sites of QUIN to the VC1 domain for both rat and human RAGE. QUIN was found to bind at multiple sites to the VC1 dimer, each leading to particular mechanistic scenarios for the signaling evoked by QUIN binding, some of which directly alter RAGE oligomerization. This work contributes to the understanding of the phenomenon of RAGE-QUIN recognition, leading to the modulation of RAGE function.     

Expression, purification and fluorine-18 radiolabeling of recombinant S100 proteins--potential probes for molecular imaging of receptor for advanced glycation endproducts (RAGE) in vivo

Data concerning the pathophysiological role of the interaction of circulating S100 proteins, a multigenic family of Ca(2+)-modulated proteins, with the receptor for advanced glycation endproducts (RAGE) in cardiovascular diseases, inflammatory processes, and tumorigenesis in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A1, S100B, and S100A12 as potential probes for molecular imaging of this interaction. Therefore, human S100 proteins were cloned as GST fusion proteins in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100 proteins were radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiolabeled recombinant S100 proteins ((18)F-S100) were used in biodistribution experiments and small animal positron emission tomography (PET) studies in rats. The tissue-specific distribution of (18)F-S100 proteins in vivo correlated well with the anatomical localization of RAGE, e.g., in lungs and in the vascular system. These findings indicate circulating S100A1, S100B, and S100A12 proteins to be ligands for RAGE in rats in vivo. The approach allows the use of small animal PET and provides novel probes to delineate functional expression of RAGE under normal and pathophysiological conditions in rodent models of disease.     

The G82S polymorphism promotes glycosylation of the receptor for advanced glycation end products (RAGE) at asparagine 81: comparison of wild-type rage with the G82S polymorphic variant

Interaction between the receptor for advanced glycation end products (RAGE) and its ligands amplifies the proinflammatory response. N-Linked glycosylation of RAGE plays an important role in the regulation of ligand binding. Two potential sites for N-linked glycosylation, at Asn(25) and Asn(81), are implicated, one of which is potentially influenced by a naturally occurring polymorphism that substitutes Gly(82) with Ser. This G82S polymorphic RAGE variant displays increased ligand binding and downstream signaling. We hypothesized that the G82S polymorphism affects RAGE glycosylation and thereby affects ligand binding. WT or various mutant forms of RAGE protein, including N25Q, N81Q, N25Q/G82S, and N25Q/N81Q, were produced by transfecting HEK293 cells. The glycosylation patterns of expressed proteins were compared. Enzymatic deglycosylation showed that WT RAGE and the G82S polymorphic variant are glycosylated to the same extent. Our data also revealed N-linked glycosylation of N25Q and N81Q mutants, suggesting that both Asn(25) and Asn(81) can be utilized for N-linked glycosylation. Using mass spectrometry analysis, we found that Asn(81) may or may not be glycosylated in WT RAGE, whereas in G82S RAGE, Asn(81) is always glycosylated. Furthermore, RAGE binding to S100B ligand is affected by Asn(81) glycosylation, with consequences for NF-κB activation. Therefore, the G82S polymorphism promotes N-linked glycosylation of Asn(81), which has implications for the structure of the ligand binding region of RAGE and might explain the enhanced function associated with the G82S polymorphic RAGE variant.     

Structural Insights into Calcium-Bound S100P and the V Domain of the RAGE Complex

The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE) and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca²⁺-bound S100P using various biophysical techniques, including isothermal titration calorimetry (ITC), fluorescence spectroscopy, multidimensional NMR spectroscopy, functional assays and site-directed mutagenesis. The entropy-driven binding between the V domain of RAGE and Ca⁺²-bound S100P was found to lie in the micromolar range (K_d of ∼6 µM). NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases.     

S100B engages RAGE or bFGF/FGFR1 in myoblasts depending on its own concentration and myoblast density. Implications for muscle regeneration

In high-density myoblast cultures S100B enhances basic fibroblast growth factor (bFGF) receptor 1 (FGFR1) signaling via binding to bFGF and blocks its canonical receptor, receptor for advanced glycation end-products (RAGE), thereby stimulating proliferation and inhibiting differentiation. Here we show that upon skeletal muscle injury S100B is released from myofibers with maximum release at day 1 post-injury in coincidence with satellite cell activation and the beginning of the myoblast proliferation phase, and declining release thereafter in coincidence with reduced myoblast proliferation and enhanced differentiation. By contrast, levels of released bFGF are remarkably low at day 1 post-injury, peak around day 5 and decline thereafter. We also show that in low-density myoblast cultures S100B binds RAGE, but not bFGF/FGFR1 thereby simultaneously stimulating proliferation via ERK1/2 and activating the myogenic program via p38 MAPK. Clearance of S100B after a 24-h treatment of low-density myoblasts results in enhanced myotube formation compared with controls as a result of increased cell numbers and activated myogenic program, whereas chronic treatment with S100B results in stimulation of proliferation and inhibition of differentiation due to a switch of the initial low-density culture to a high-density culture. However, at relatively high doses, S100B stimulates the mitogenic bFGF/FGFR1 signaling in low-density myoblasts, provided bFGF is present. We propose that S100B is a danger signal released from injured muscles that participates in skeletal muscle regeneration by activating the promyogenic RAGE or the mitogenic bFGF/FGFR1 depending on its own concentration, the absence or presence of bFGF, and myoblast density.     

S100 to receptor for advanced glycation end-products binding assay: Looking for inhibitors

Secreted by tumor and stromal cells, S100 proteins exert their biological functions via the interaction with surface receptors. The most described receptor is the receptor for advanced glycation end-products (RAGE), thereby participating in the S100-dependent cell migration, invasion, tumor growth, angiogenesis and metastasis. Several approaches have been described for determining this interaction. Here we describe an easy, specific and highly reproducible ELISA-based method, by optimizing several parameters such as the binding and blocking buffer, interaction time and concentrations, directed to screen chemical and biological inhibitors of this interaction for S100A4, S100A7 and S100P proteins. The efficiency of the protocol was validated by using well described neutralizing agents of the RAGE receptor and of the S100A4 activity. The methodology described here will allow future works with other members of the S100 protein family and their receptors.     

Chemotactic activity of S100A7 (Psoriasin) is mediated by the receptor for advanced glycation end products and potentiates inflammation with highly homologous but functionally distinct S100A15

Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.