Investigation of the Ca2+-dependent interaction of trifluoperazine with S100a: A19F NMR and circular dichroism study

S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca²⁺-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca²⁺, its interaction with trifluoperazine (TFP) was investigated using¹⁶F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K _d) values of TFP, as estimated from the chemical shifts of¹⁹F NMR, were 191 and 29 μm in the absence and presence of Ca²⁺, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca²⁺-bound S100a was 65 Hz greater than in the presence of Ca²⁺-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca²⁺-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca²⁺-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca²⁺ induces a small transition from α-helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca²⁺-bound S100a; this observation is similar to the effect of TFP on Ca²⁺-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.     

Investigation of the Ca2+-dependent interaction of trifluoperazine with S100a: A19F NMR and circular dichroism study

S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca²⁺-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca²⁺, its interaction with trifluoperazine (TFP) was investigated using¹⁶F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K _d) values of TFP, as estimated from the chemical shifts of¹⁹F NMR, were 191 and 29 m in the absence and presence of Ca²⁺, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca²⁺-bound S100a was 65 Hz greater than in the presence of Ca²⁺-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca²⁺-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca²⁺-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca²⁺ induces a small transition from -helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca²⁺-bound S100a; this observation is similar to the effect of TFP on Ca²⁺-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.     

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.     

Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca²⁺-dependent manner. In order to study the Ca²⁺-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [¹⁹F]- and [¹H]-NMR and UV-difference spectroscopy. It was estimated from [¹⁹F]-NMR that in the absence of Ca²⁺, thek _1/2 value of TFP was 130 µM, while itsk _1/2 value decreased to 28 µM in the presence of Ca²⁺. The addition of KCl was not antagonistic to the Ca²⁺-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca²⁺-bound S100b was estimated to be 9×10² sec^–1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca²⁺ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg²⁺ was added to a S100b-TFP solution. Thus, we suggest that Ca²⁺ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.     

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

CaBP4 modulates Ca²⁺-dependent activity of L-type voltage-gated Ca²⁺ channels (Cav1.4) in retinal photoreceptor cells. Mg²⁺ binds to the first and third EF-hands (EF1 and EF3), and Ca²⁺ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg²⁺-bound and Ca²⁺-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1–99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg²⁺ or Ca²⁺ bound at EF1. The C-lobe binds Ca²⁺ at EF3 and EF4 and exhibits a Ca²⁺-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca²⁺-bound CaBP4 (Phe¹³⁷, Glu¹⁶⁸, Leu²⁰⁷, Phe²¹⁴, Met²⁵¹, Phe²⁶⁴, and Leu²⁶⁸) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca²⁺-dependent inactivation domain.     

Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca²⁺-dependent manner. In order to study the Ca²⁺-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [¹⁹F]- and [¹H]-NMR and UV-difference spectroscopy. It was estimated from [¹⁹F]-NMR that in the absence of Ca²⁺, thek _1/2 value of TFP was 130 µM, while itsk _1/2 value decreased to 28 µM in the presence of Ca²⁺. The addition of KCl was not antagonistic to the Ca²⁺-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca²⁺-bound S100b was estimated to be 9×10² sec^?1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca²⁺ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg²⁺ was added to a S100b-TFP solution. Thus, we suggest that Ca²⁺ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.     

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY*?

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca²⁺-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca²⁺-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca²⁺-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a “fuzzy” complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.     

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.     

Long-Timescale Molecular Dynamics Simulations Elucidate the Dynamics and Kinetics of Exposure of the Hydrophobic Patch in Troponin C

Troponin (Tn) is an important regulatory protein in the thin-filament complex of cardiomyocytes. Calcium binding to the troponin C (TnC) subunit causes a change in its dynamics that leads to the transient opening of a hydrophobic patch on TnC’s surface, to which a helix of another subunit, troponin I (TnI), binds. This process initiates contraction, making it an important target for studies investigating the detailed molecular processes that underlie contraction. Here we use microsecond-timescale Anton molecular dynamics simulations to investigate the dynamics and kinetics of the opening transition of the TnC hydrophobic patch. Free-energy differences for opening are calculated for wild-type Ca²⁺-bound TnC (∼8 kcal/mol), V44Q Ca²⁺-bound TnC (3.2 kcal/mol), E40A Ca²⁺-bound TnC (∼12 kcal/mol), and wild-type apo TnC (∼20 kcal/mol). These results suggest that the mutations have a profound impact on the frequency with which the hydrophobic patch presents to TnI. In addition, these simulations corroborate that cardiac wild-type TnC does not open on timescales relevant to contraction without calcium being bound.     

Energetics and protomer communication in the dynamical structure of S100A13 in free and protein-bound states

S100A13 is S100 family of EF-hand-containing calcium-binding protein involved in the secretion of some growth factors and pro-inflammatory cytokines lacking signal peptides. The involvement of S100A13 in cancer progression and inflammatory diseases has been reported. In this study, structures generated during atomistic molecular dynamics simulation were studied. Dynamical network analysis data revealed that native inter-protomer communication network driven principally by vdW interaction (~550 kj/mol) is altered (Receptor for advanced glycation end products (RAGE) C2- and Fibroblast growth factor (FGF)-1-bound S100A13) or completely abolished (interleukin-1 (IL1)-α- and C2A-p40Syt1-bound S100A13) in protein-bound S100A13 homodimer. Bulk water density (weighted atomic density) around exposed S100A13 homodimer surface explored tends to follow the dynamical network lead as S100A13 homodimer appeared densely solvated in C2A-p40Syt1- and IL1)-α-bound states but not in RAGE C2- and FGF-1-bound biosystems. Furthermore, projection of radius of gyration and root mean square deviation (from native structure) variables of the generated structures along the 3D-free energy surface showed anti-parallel β-sheet proximal to Ca²⁺-binding loops-I/II in most metastable complexes retrieved from energy minima state with strong indications for β-sheet network formation during protein binding. Interaction between S100A13 homodimer and ligand–proteins may be dictated by the strength of vdW and electrostatic interaction with possible involvement of bulk water desolvation in some complexes. All these results strongly suggest that disruption of multiprotein receptor complex can be achieved by designing specific compounds targeting a specific aspect of S100A13/protein interaction; such drugs may have clinical usefulness in blocking angiogenesis, reversing cell proliferation and attenuating inflammatory processes.     

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca²⁺-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1^V77E) in the Ca²⁺-free/Mg²⁺-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca²⁺-dependent differences for residues 170–174. An NMR-derived model of GCAP1^V77E contains Mg²⁺ bound at EF2 and looks similar to Ca²⁺ saturated GCAP1 (root mean square deviations = 2.0 Å). Ca²⁺-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164–174 called the Ca²⁺ switch helix). Ca²⁺-induced shortening of the Ca²⁺ switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca²⁺ switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca²⁺ binding affinity and abolished RetGC1 activation. We conclude that Ca²⁺-dependent conformational changes in the Ca²⁺ switch helix are important for activating RetGC1 and provide further support for a Ca²⁺-myristoyl tug mechanism.     

Structural Insights for Activation of Retinal Guanylate Cyclase by GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca²⁺-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca²⁺-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca²⁺-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca²⁺ binding in EF-hand 4 and models the Ca²⁺-free/Mg²⁺-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca²⁺-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca²⁺-free activator and Ca²⁺-bound inhibitor states. This contrasts with large Ca²⁺-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1.     

Solution Structure of S100A1 Bound to the CapZ Peptide (TRTK12)

As is typical for S100-target protein interactions, a Ca²⁺-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping protein CapZ. In addition, the Ca²⁺-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca²⁺-loaded S100A1. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca²⁺-bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e., S100A1-RyRP12), which illustrated how the binding pocket in Ca²⁺-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when the structures of S100A1-TRTK12 and S100B-TRTK12 were compared, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those with other S100-target and S100-drug complexes, provide the basis for designing novel small-molecule inhibitors that could be specific for blocking one or more S100-target protein interactions.     

The Effects of CapZ Peptide (TRTK-12) Binding to S100B-Ca as Examined by NMR and X-ray Crystallography

Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca²⁺-S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca²⁺-S100B (1.5-Å resolution) and S100B-Ca²⁺-TRTK-12 (2.0-Å resolution) determined here indicate that the S100B-Ca²⁺-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca²⁺-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca²⁺-p53 peptide complex, TRTK-12 binding to Ca²⁺-S100B was found to increase the protein's Ca²⁺-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca²⁺ ligands and/or improved the Ca²⁺ coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca²⁺-TRTK-12 and S100B-Ca²⁺ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD = 0.19). On the other hand, B-factors for residues in EF2 of Ca²⁺-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR ¹⁵N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca²⁺-S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca²⁺-S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.     

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7: A REGULATOR OF GOLGI TRAFFICKING*

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca²⁺ binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca²⁺ but not Mg²⁺ and undergoes significant conformational changes in both secondary and tertiary structure upon Ca²⁺ binding. The Ca²⁺-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca²⁺-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.     

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.     

Ca-modulated ONE-GC odorant signal transduction

In a subset of olfactory epithelium the odorant receptor guanylate cyclase, ONE-GC, is a central transduction component of the cyclic GMP signaling pathway. The odorant binds to the extracellular domain and activates its intracellular catalytic domain to generate the odorant second messenger, cyclic GMP. The present study demonstrates that it is a two-step, Ca²⁺-independent and Ca²⁺-dependent, sequential process. In step one, the odorant, uroguanylin, binds ONE-GC and primes it for stimulation. In step two, Ca²⁺-bound neurocalcin δ binds to the defined intracellular domain and saturates ONE-GC activity. A prototype model is proposed that depicts this signal transduction process.     

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.     

Mechanism of the Ca-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca²⁺ binds to the EF2 domain of S100A4 with micromolar affinity and that the K_d value for Ca²⁺ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K_d results from a reduced dissociation rate constant (from 16 s^− 1 to 0.3 s^− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca²⁺]. However, for Ca²⁺ transients to be effective in further promoting dissociation, the elevated Ca²⁺ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.