In silico fragment-based discovery of CIB1-directed anti-tumor agents by FRASE-bot.

Chemical probes are an indispensable tool for translating biological discoveries into new therapies, though are increasingly difficult to identify since novel therapeutic targets are often hard-to-drug proteins. We introduce FRASE-based hit-finding robot (FRASE-bot), to expedite drug discovery for unconventional therapeutic targets. FRASE-bot mines available 3D structures of ligand-protein complexes to create a database of FRAgments in Structural Environments (FRASE). The FRASE database can be screened to identify structural environments similar to those in the target protein and seed the target structure with relevant ligand fragments. A neural network model is used to retain fragments with the highest likelihood of being native binders. The seeded fragments then inform ultra-large-scale virtual screening of commercially available compounds. We apply FRASE-bot to identify ligands for Calcium and Integrin Binding protein 1 (CIB1), a promising drug target implicated in triple negative breast cancer. FRASE-based virtual screening identifies a small-molecule CIB1 ligand (with binding confirmed in a TR-FRET assay) showing specific cell-killing activity in CIB1-dependent cancer cells, but not in CIB1-depletion-insensitive cells.

Biophysical and structural studies of fibulin-2.

Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.

Structural and molecular dynamics simulation studies of CBL-interacting protein kinase CIPK and its complexes related to plant salinity stress.

CONTEXT: Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na+-to-K+ ratio. The CBL-CIPK interaction is pivotal for Ca2+-dependent salinity stress signaling. METHODS: Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein-protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500 ns and 300 ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300 ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

Formation of the calpain-1/calpastatin complex promotes activation of calpain-1 under oxidizing conditions.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

Protein degradation in skeletal muscle is a key component of protein turnover and maintenance of muscle function. Protein degradation in postmortem muscle is commonly observed and is associated with the accumulation of degradation products and improved meat tenderness. Because there is significant evidence that calpain-1 is involved with proteolysis of muscle proteins in both situations, defining the factors that regulate calpain activity will position scientists to improve calpain-1 activity in both contexts. Calpain-1 is a neutral calcium-dependent proteinase that is inhibited by calpastatin, oxidation, and slightly acidic pH environments. Because oxidation of the calpain/calpastatin complex with hydrogen peroxide appeared to activate calpain-1, we hypothesize that calpastatin binding to calpain may protect the active site cysteine. In the current study, we tested this hypothesis and investigated how n-ethyl maleimide (NEM), an alkylating agent, affects the regulation of calpain in the presence and absence of calpastatin molecules. The results suggest that calpastatin can protect calpain-1 from reacting with maleimide-polyethylene glycol but that exposure of calpain-1/calpastatin complex to NEM or hydrogen peroxide resulted in autolysis and activation of calpain. Under some circumstances, calpastatin appears to protect calpain-1 from inhibition by modification of active site cysteine. These novel observations show a different role for calpastatin and give reason to interpret calpastatin abundance and activity data in a different light.

Deciphering the dual nature of nesfatin-1: a tale of zinc ion's Janus-faced influence.

BACKGROUND: Nucleobindin-2 (Nucb2) and nesfatin-1 (N1) are widely distributed hormones that regulate numerous physiological processes, from energy homeostasis to carcinogenesis. However, the role of nesfatin-2 (N2), the second product of Nucb2 proteolytic processing, remains elusive. To elucidate the relationship between the structure and function of nesfatins, we investigated the properties of chicken and human homologs of N1, as well as a fragment of Nucb2 consisting of N1 and N2 conjoined in a head-to-tail manner (N1/2). RESULTS: Our findings indicate that Zn(II) sensing, in the case of N1, is conserved between chicken and human species. However, the data presented here reveal significant differences in the molecular features of the analyzed peptides, particularly in the presence of Zn(II). We demonstrated that Zn(II) has a Janus effect on the M30 region (a crucial anorexigenic core) of N1 and N1/2. In N1 homologs, Zn(II) binding results in the concealment of the M30 region driven by a disorder-to-order transition and adoption of the amyloid fold. In contrast, in N1/2 molecules, Zn(II) binding causes the exposure of the M30 region and its destabilization, resulting in strong exposure of the region recognized by prohormone convertases within the N1/2 molecule. CONCLUSIONS: In conclusion, we found that Zn(II) binding is conserved between chicken and human N1. However, despite the high homology of chicken and human N1, their interaction modes with Zn(II) appear to differ. Furthermore, Zn(II) binding might be essential for regulating the function of nesfatins by spatiotemporally hindering the N1 anorexigenic M30 core and concomitantly facilitating N1 release from Nucb2.

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Isolation and differential structure characteristics of calcium-binding peptides derived from Pacific cod bones by hydroxyapatite affinity.

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 µg/mg to 7.94 ± 1.56 µg/mg. Peptides with a molecular weight of 1-2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to ß-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide­calcium complexes. The F21 contained 42 peptides, with repeated "GE" motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides.

1H, 13C, and 15N resonance assignments and solution structure of the N-terminal divergent calponin homology (NN-CH) domain of human intraflagellar transport protein 54.

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.

Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Uncovering allostery and regulation in SORCIN through molecular dynamics simulations.

Soluble resistance-related calcium-binding protein or Sorcin is an allosteric, calcium-binding Penta-EF hand (PEF) family protein implicated in multi-drug resistant cancers. Sorcin is known to bind chemotherapeutic molecules such as Doxorubicin. This study uses in-silico molecular dynamics simulations to explore the dynamics and allosteric behavior of Sorcin in the context of Ca2+ uptake and Doxorubicin binding. The results show that Ca2+ binding induces large, but reversible conformational changes in the Sorcin structure which manifest as rigid body reorientations that preserve the local secondary structure. A reciprocal allosteric handshake centered around the EF5 hand is found to be key in Sorcin dimer formation and stabilization. Binding of Doxorubicin results in rearrangement of allosteric communities which disrupts long-range allosteric information transfer from the N-terminal domain to the middle lobe. However, this binding does not result in secondary structure destabilization. Sorcin does not appear to have a distinct Ca2+ activated mode of Doxorubicin binding.Communicated by Ramaswamy H. Sarma.

Development of a fluorescent multiplexed lateral flow immunoassay for the simultaneous detection of crustacean allergen tropomyosin, sarcoplasmic calcium binding protein and egg allergen ovalbumin in different matrices and commercial foods.

A quantum dot (QD) based multiplexed lateral flow immunoassay (xLFIA) for the simultaneous detection of egg allergen ovalbumin, crustacean allergen tropomyosin (TM) and sarcoplasmic calcium binding protein (SCP) was developed in this study. QD-labeled rabbit anti-ovalbumin, SCP and TM antibodies were applied as fluorescent detection probes. The chromatography system was optimized to reduce the mutual interference of different test lines. Visual and instrumental detection limits of the xLFIA were 0.1 and 0.05 µg/mL for SCP, both 0.05 µg/mL for ovalbumin and both 0.5 µg/mL for TM. As low as 0.10 % crab powder, 0.01 % egg white powder and 0.05 % shrimp powder could be detected in all three model foods using xLFIA. Besides, the xLFIA detection results of 23 of 28 commercial foods were consistent with ingredient labels. These findings indicate that the developed xLFIA is a practical tool for point-of-care detection of egg and crustacean allergens in processed and commercial foods.

Machine learning-based modulation of Ca2+-binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding.

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca2+ with higher affinity than Wt-Nt-EhCaBP1, by ∼600 times. The crystal structure of the mutant displayed more compact Ca2+-coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca2+ even in another EF-hand loop.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Purification, Expression, and Characterization of Sarcoplasmic Calcium Binding Protein: A Novel Allergen of Portunus trituberculatus.

Swimming crab (Portunus trituberculatus), a crucial valuable crustacean, is a common factor causing food allergy. However, studies on allergens of P. trituberculatus are scarce. In this study, the sarcoplasmic calcium binding protein (SCP) of P. trituberculatus was expressed in Escherichia coli, purified with affinity chromatography, and the IgE-binding activity was evaluated through serological analyses. Further, the structure, physicochemical properties, and cross-reactivity were assessed via bioinformatics, immunologic, and spectroscopy techniques. The results indicated that P. trituberculatus SCP was an allergen displaying strong IgE-binding capacity, composed of 60% α-helix. It presented good immunologic and structural stability at 4-70 °C and pH 3-10, and only exhibited high IgG cross-reactivity among crustaceans, without cross-reactivity with other species tested. These results establish the foundations for further studies on SCP and are promising to promote the development of specific crustacean allergen detection and precise allergy diagnosis.

Functional Role of an Unusual Transmembrane Acidic Residue in the Calcium Pump Regulator Myoregulin.

Myoregulin (MLN) is a member of the regulin family, a group of homologous membrane proteins that bind to and regulate the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). MLN, which is expressed in skeletal muscle, contains an acidic residue in its transmembrane domain. The location of this residue, Asp35, is unusual because the relative occurrence of aspartate is very rare (<0.2%) within the transmembrane helix regions. Therefore, we used atomistic simulations and ATPase activity assays of protein co-reconstitutions to probe the functional role of MLN residue Asp35. These structural and functional studies showed Asp35 has no effects on SERCA's affinity for Ca2+ or the structural integrity of MLN in the lipid bilayer. Instead, Asp35 controls SERCA inhibition by populating a bound-like orientation of MLN. We propose Asp35 provides a functional advantage over other members of the regulin family by populating preexisting MLN conformations required for MLN-specific regulation of SERCA. Overall, this study provides new clues about the evolution and functional divergence of the regulin family and offers novel insights into the functional role of acidic residues in transmembrane protein domains.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis.

Ca2+-dependent activator proteins for secretion (CAPSs) are required for Ca2+-regulated exocytosis in neurons and neuroendocrine cells. CAPSs contain a pleckstrin homology (PH) domain that binds PI(4,5)P2-membrane. There is also a C2 domain residing adjacent to the PH domain, but its function remains unclear. In this study, we solved the crystal structure of the CAPS-1 C2PH module. The structure showed that the C2 and PH tandem packs against one another mainly via hydrophobic residues. With this interaction, the C2PH module exhibited enhanced binding to PI(4,5)P2-membrane compared with the isolated PH domain. In addition, we identified a new PI(4,5)P2-binding site on the C2 domain. Disruption of either the tight interaction between the C2 and PH domains or the PI(4,5)P2-binding sites on both domains significantly impairs CAPS-1 function in Ca2+-regulated exocytosis at the Caenorhabditis elegans neuromuscular junction (NMJ). These results suggest that the C2 and PH domains constitute an effective unit to promote Ca2+-regulated exocytosis.

Hysteretic Pressure Dependence of Ca2+ Binding in LH1 Bacterial Membrane Chromoproteins.

Much of the thermodynamic parameter values that support life are set by the properties of proteins. While the denaturing effects of pressure and temperature on proteins are well documented, their precise structural nature is rarely revealed. This work investigates the destabilization of multiple Ca2+ binding sites in the cyclic LH1 light-harvesting membrane chromoprotein complexes from two Ca-containing sulfur purple bacteria by hydrostatic high-pressure perturbation spectroscopy. The native (Ca-saturated) and denatured (Ca-depleted) phases of these complexes are well distinguishable by much-shifted bacteriochlorophyll a exciton absorption bands serving as innate optical probes in this study. The pressure-induced denaturation of the complexes related to the failure of the protein Ca-binding pockets and the concomitant breakage of hydrogen bonds between the pigment chromophores and protein environment were found cooperative, involving all or most of the Ca2+ binding sites, but irreversible. The strong hysteresis observed in the spectral and kinetic characteristics of phase transitions along the compression and decompression pathways implies asymmetry in the relevant free energy landscapes and activation free energy distributions. A phase transition pressure equal to about 1.9 kbar was evaluated for the complexes from Thiorhodovibrio strain 970 from the pressure dependence of biphasic kinetics observed in the minutes to 100 h time range.

Crystal Structure Analysis of Sarcoplasmic-Calcium-Binding Protein: An Allergen in Scylla paramamosain.

The structure of allergenic proteins provides important information about the binding of allergens to antibodies. In this study, the crystal structure of Scy p 4 with a resolution of 1.60 Å was obtained by X-ray diffraction. Epitope mapping of Scy p 4 revealed that linear epitopes are located on the surface of Scy p 4. Also, conformational epitopes are mostly located in the structural conservative region. Further structural comparison, surface electrostatic potential, and hydrogen bond force analysis showed that mutation of Asp70 and Asp18/20/70 would lead to calcium-binding capacity being lost and destruction of allergenicity. Furthermore, a comparative analysis of structure showed that sarcoplasmic-calcium-binding protein (SCP) had high sequence, secondary, and spatial structural identity in crustaceans, which may be an important factor leading to cross-reactivity among crustaceans. The structure of Scy p 4 provides a template for epitope evaluation and localization of SCPs, which will help to reveal cross-reactivity among species.