Copper induces the assembly of a multiprotein aggregate implicated in the release of fibroblast growth factor 1 in response to stress.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu2+-binding protein and Cu2+ is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu2+ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity and to interact in the presence of Cu2+ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu2+ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu2+ with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu2+ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu2+ oxidation may be an essential post-translational intracellular modifier of this process.     

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.     

S100A9 gene silencing inhibits the release of pro‐inflammatory cytokines by blocking the IL‐17 signalling pathway in mice with acute pancreatitis

The study aimed to investigate whether S100A9 gene silencing mediating the IL‐17 pathway affected the release of pro‐inflammatory cytokines in acute pancreatitis (AP). Kunming mice were assigned to the normal, AP, AP + negative control (NC), AP + shRNA, AP + IgG and AP + anti IL‐17 groups. ELISA was applied to measure expressions of AMY, LDH, CRP, TNF‐α, IL‐6 and IL‐8. The cells were distributed into the control, blank, NC, shRNA1 and shRNA2 groups. MTT assay, flow cytometry, RT‐qPCR and Western blotting were used to evaluate cell proliferation, cell cycle and apoptosis, and expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12 in tissues and cells. Compared with the normal group, the AP group displayed increased expressions of AMY, LDH, CRP, TNFα, IL‐6, IL‐8, S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12. The AP + shRNA and AP + anti IL‐17 groups exhibited an opposite trend. The in vivo results: Compare with the control group, the blank, NC, shRNA1 and shRNA2 groups demonstrated increased expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12, as well as cell apoptosis and cells at the G1 phase, with reduced proliferation. Compared with the blank and NC groups, the shRNA1 and shRNA2 groups had declined expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12, as well as cell apoptosis and cells at the G1 phase, with elevated proliferation. The results indicated that S100A9 gene silencing suppressed the release of pro‐inflammatory cytokines through blocking of the IL‐17 pathway in AP.     

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.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.     

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE)

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.     

Crystal structure study on human S100A13 at 2.0 A resolution

The S100 protein family is the largest group of calcium-binding protein families, which consists of at least 25 members. S100A13, which is widely expressed in a variety of tissues, is a unique member of the S100 protein family. Previous reports showed that S100A13 might be involved in the stress-induced release of some signal peptide-less proteins (such as FGF-1 and IL-1alpha) and also associated with inflammatory functions. It was also reported that S100A13 is a new angiogenesis marker. Here we report the crystal structure of the Ca(2+)-bound form of S100A13 at 2.0 A resolution. S100A13 is a homodimer with four EF-hand motifs in an asymmetric unit, displaying a folding pattern similar to other S100 members. However, S100A13 has the unique structural feature with all alpha-helices being amphiphilic, which was not found in other members of S100s. We propose that this characteristic structure of S100A13 might be related to its ability to mediate the release of FGF-1 and IL-1alpha.     

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.     

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.     

Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.     

SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

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.     

钙结合蛋白S100A14的结构预测及分析

钙结合蛋白S100A14是S100家族中的新成员,其空间结构与功能尚未阐明。采用服务器PredictProtein对人S100A14进行二级结构预测,利用同源建模法构建S100A14（序列12-102）的空间结构模型,经PROCHECK评估模型的可靠性,并将所构建的单体模型进行分子对接,预测S100A14形成同源二聚体的可能性及模式。结果显示,S100A14与S100A13的蛋白序列一致性最高,其C-端Ca2＋结合区存在多个变异,但Cu2＋和Zn2＋结合位点保守存在;helix I与helix IV较S100A13延伸长,而helix I、helix II和helix IV与S100A13的四个α螺旋一样具有两亲性的结构特征,并且在S100A13中扮演重要角色的W77在S100A14的helix IV（W85）中也保守存在。空间结构上,S100A14与S100A13具极大相似性;分子对接显示S100A14单体间可以通过疏水作用力形成＂X-型螺旋束＂同源二聚体。这些结构特征的分析将为S100A14的功能研究提供重要线索。     

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.     

Human S100A12: a novel key player in inflammation?

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Human S100A12 is predominantly expressed and secreted by neutrophil granulocytes and, therefore, has been assigned to the S100 protein subfamily of calgranulins or myeloid-related proteins. Intracellular S100A12 exists as an anti-parallel homodimer and upon calcium-dependent activation interacts with target proteins to regulate cellular functions. Extracellular S100A12 exists majorily as homodimer and hexamer, respectively, and shows cytokine-like characteristics. It is part of the innate immune response and linked to certain autoimmune reactions. Human S100A12 is markedly overexpressed in inflammatory compartments, and elevated serum levels of S100A12 are found in patients suffering from various inflammatory, neurodegenerative, metabolic, and neoplastic disorders. In this regard, interaction of calcium-activated S100A12 with the multiligand receptor for advanced glycation endproducts (RAGE) and its soluble form (sRAGE) plays a central pathogenetic role. Recent clinical evidence suggests a high potential of S100A12 as a sensitive and specific diagnostic marker of localized inflammatory processes.     

Dimers of human β-defensins and their interactions with the POPG membrane

The stable dimeric structures of human β-defensin (HBD)-3 and -28 have been first computationally identified via a protein docking approach in conjunction with all-atom molecular dynamic simulation. We found that both HBD dimers contain an extended β-sheet platform stabilised mainly by the interaction of second β-sheets and further investigated interaction mechanisms of these dimers including HBD-2 against 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol membrane bilayer by using coarse-grained model combined with the ElNeDyn network. The extended β-sheet platform of the HBD dimer stayed over the bilayer due to the attachment of the amphipathic region located on one side of the β-sheet platform. The hydrophobic residues of HBDs on the surface interact with the hydrophobic tails of the lipids, whereas the positively charged residues interact with the lipid polar head groups. Finally, antimicrobial nature of HBD-2, HBD-3 and HBD-28 dimers is found to be kept because they are not detached in interacting with the membrane.     

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.     

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.     

Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor

Acidic fibroblast growth factor (aFGF) is a signal peptide-less protein that is secreted into the extracellular compartment as part of a multiprotein release complex, consisting of aFGF, S100A13 (a calcium binding protein), and a 40 kDa (p40) form of synaptotagmin (Syt1), a protein that participates in the docking of a variety of secretory vesicles. p40 Syt1, and specifically its C2A domain, is believed to play a major role in the non-classical secretion of the aFGF release complex mediated by the interaction of aFGF and p40 Syt1with the phospholipids of the cell membrane inner leaflet. In the present study, we investigate the structural characteristics of aFGF and the C2A domain of p40 Syt1 under acidic conditions, using a variety of biophysical techniques including multidimensional NMR spectroscopy. Urea-induced equilibrium unfolding (at pH 3.4) of both aFGF and the C2A domain are non-cooperative and proceed with the accumulation of stable intermediate states. 1-Anilino-8-napthalene sulfonate (ANS) binding and size-exclusion chromatography results suggest that both aFGF and the C2A domain exist as partially structured states under acidic conditions (pH 3.4). Limited trypsin digestion analysis and 1H-15N chemical shift perturbation data reveal that the flexibility of certain portions of the protein backbone is increased in the partially structured state(s) of aFGF. The residues that are perturbed in the partially structured state(s) in aFGF are mostly located at the N- and C-terminal ends of the protein. In marked contrast, most of the interactions stabilizing the native secondary structure are preserved in the partially structured state of the C2A domain. Isothermal titration calorimetry data indicate that the binding affinity between aFGF and the C2A domain is significantly enhanced at pH 3.4. In addition, both aFGF and the C2A domain exhibit much higher lipid binding affinity in their partially structured states. The translocation of the multiprotein FGF release complex across the membrane appears to be facilitated by the formation of partially structured states of aFGF and the C2A domain of p40 Syt1.     

Structures and free energy landscapes of aqueous zinc(II)-bound amyloid-β(1–40) and zinc(II)-bound amyloid-β(1–42) with dynamics

Binding of divalent metal ions with intrinsically disordered fibrillogenic proteins, such as amyloid-β (Aβ), influences the aggregation process and the severity of neurodegenerative diseases. The Aβ monomers and oligomers are the building blocks of the aggregates. In this work, we report the structures and free energy landscapes of the monomeric zinc(II)-bound Aβ40 (Zn:Aβ40) and zinc(II)-bound Aβ42 (Zn:Aβ42) intrinsically disordered fibrillogenic metallopeptides in an aqueous solution by utilizing an approach that employs first principles calculations and parallel tempering molecular dynamics simulations. The structural and thermodynamic properties, including the secondary and tertiary structures and conformational Gibbs free energies of these intrinsically disordered metallopeptide alloforms, are presented. The results show distinct differing characteristics for these metallopeptides. For example, prominent β-sheet formation in the N-terminal region (Asp1, Arg5, and Tyr10) of Zn:Aβ40 is significantly decreased or lacking in Zn:Aβ42. Our findings indicate that blocking multiple reactive residues forming abundant β-sheet structure located in the central hydrophobic core and C-terminal regions of Zn:Aβ42 via antibodies or small organic molecules might help to reduce the aggregation of Zn(II)-bound Aβ42. Furthermore, we find that helix formation increases but β-sheet formation decreases in the C-terminal region upon Zn(II) binding to Aβ. This depressed β-sheet formation in the C-terminal region (Gly33-Gly38) in monomeric Zn:Aβ42 might be linked to the formation of amorphous instead of fibrillar aggregates of Zn:Aβ42.