Calcium Binding Sites Research Articles

Polyphenol-treatment without preceding glutaraldehyde fixation: a potential paradigm change for bioprosthetic heart valves

Abstract Background Glutaraldehyde (GA) is the common fixative agent for bioprosthetic heart valves (BHVs). However, its chemical instability can expose calcium binding sites and, at commercially used concentrations, GA does not ensure effective immunological tolerance, triggering an immune response correlated to degeneration. Recently, polyphenols (PPs) achieved an almost complete xenoantigens inactivation in GA-fixed BHVs ensuring chemical stability of the treated tissue while improving mechanical characteristics and adding anti-infective and anti-thrombotic properties [1-4]. Purpose A new PP-based treatment has been introduced as a potential GA substitute in BHV manufacturing. The unique chemical properties of PPs permit them to interact with the tissue through stable chemical bonds without depolymerization over time, achieving several improvements that promise to extend the long-term durability of BHVs (Fig.1). Methods The study compared the effects of two cross-linking methods on bovine pericardial tissue: (1) traditional fixation in GA, and (2) treatment with PPs without GA. The type of chemical interaction between PPs and tissue was evaluated through nuclear magnetic resonance. The xenoantigens inactivation was quantified in-vitro by ELISA test and in-vivo in a humanized mouse animal model. Biomechanical properties were assessed through uniaxial stress-strain tests and cross-link density through the shrinkage temperature test. The protective effect against bacterial adhesion was evaluated against a strain of S aureus, while the inhibition of thrombosis was evaluated in-vivo in a pig animal model and in-vitro in a blood-loop using bovine blood with radio-labeled platelets. Results PPs effectuated multiple chemical bonds with and within the pericardium including cross-links based on covalent and hydrogen bonds, stacking, and electrostatic interactions. The inactivation of over 90% of surface-xenoantigens of the treated tissue allowed for superior biocompatibility (compared to <50% by commercial GA concentrations). The increase in elongation exhibited by PP-treated tissue confirmed excellent biomechanical features (Fig.2) while a sufficient degree of cross-linking was demonstrated through an adequate shrinkage temperature. Furthermore, a significant degree of bacterial adhesion (86% inhibition evaluated with S. aureus) and thrombus formation (Fig.2, up to 90% inhibition) were observed. Conclusion There is a real clinical need for improved chemical stability, calcification resistance, and immunological tolerance of current BHVs (surgical and transcatheter). PP technology may substitute the use of GA extending their performance and durability, thereby opening trans-catheter heart valves to younger patients. Reducing the need for mechanical heart valves would also address the associated risks of lifelong anticoagulation therapy.Figure 1Figure 2

Initiation of Medial Calcification: Revisiting Calcium Ion Binding to Elastin.

Pathological calcification of elastin, a key connective tissue protein in the medial layers of blood vessels, starts with the binding of calcium ions. This Mini-Review focuses on understanding how calcium ions interact with elastin to initiate calcification at a molecular level, and emphasizes water's critical role in mediating this interaction. In the past decade, great strides have been made in understanding and modeling ion-specific hydration and its effects on biomolecule interactions. However, these advances have been largely absent from our understanding of elastin calcification. Historically, charge-neutral backbone carbonyls and negatively charged carboxyl groups have been proposed as elastin's calcium binding sites. Recently, tropoelastin's only four carboxyl groups have been identified as binding sites from classical molecular dynamics (MD). While carboxyl groups have a much higher affinity for binding calcium ions than backbone carbonyls, conflicting evidence persists for both functional group's importance in elastin calcification. This can be attributed to the fact that divalent ions strongly polarize water, leading to a hydration shell that shields electrostatic forces. The hydration shell surrounding both a calcium ion and either of the proposed binding sites must be displaced to enable binding. Providing our own extended X-ray absorption fine structure (EXAFS) data and complementary simulations, we discuss the potential structures of calcium binding in elastin and review prior knowledge regarding the relative importance of the two proposed binding sites.

Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus

Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0–10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba2+, Co2+, and Cu2+ showed inhibitory effects on the activity of LysVPB, while Ca2+ can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.

Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity.

A wide variety of factors influence inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) activity resulting in modulation of intracellular Ca 2+ release. This regulation is thought to define the spatio-temporal patterns of Ca 2+ signals necessary for the appropriate activation of downstream effectors. The binding of both IP 3 and Ca 2+ are obligatory for IP 3 R channel opening, however, Ca 2+ regulates IP 3 R activity in a biphasic manner. Mutational studies have revealed that Ca 2+ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP 3 R channel opening without altering the Ca 2+ dependency for channel inactivation. These data suggest a distinct low-affinity Ca 2+ binding site is responsible for the reduction in IP 3 R activity at higher [Ca 2+ ]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca 2+ in cryo-EM structures of the IP 3 R type 3. This site is termed the "CD Ca 2+ binding site" and is well-conserved in all IP 3 R sub-types. We show that the CD site Ca 2+ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP 3 -generating agonists. Ca 2+ binding mutants displayed spontaneous elemental Ca 2+ events (Ca 2+ puffs) and the number of IP 3 -induced Ca 2+ puffs was significantly augmented in cells stably expressing Ca 2+ binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca 2+ ] on single channel-open probability (P o ) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca 2+ binding to the putative CD Ca 2+ inhibitory site facilitates the reduction in IP 3 R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca 2+ binding motifs may contribute to the biphasic regulation of IP 3 -induced Ca 2+ release.

Novel interplay between agonist and calcium binding sites modulates drug potentiation of α7 acetylcholine receptor.

Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drugmodulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium mayalter this balance and modulate the ability of PNU to potentiate α7.

Identification of a magnesium-binding site at the primary allosteric calcium sensor of the sodium-calcium exchanger: Implications for physiological regulation.

Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.

Rare Earth Element Binding and Recovery by a Beta Roll-Forming RTX Domain.

The Block V of the RTX domain of the adenylate cyclase protein from Bordetella pertussis is disordered, and upon binding eight calcium ions, it folds into a beta roll domain with a C-terminal capping group. Due to their similar ionic radii and coordination geometries, trivalent lanthanide ions have been used to probe and identify calcium-binding sites in many proteins. Here, we report using a FRET-based assay that the RTX domain can bind rare earth elements (REEs) with higher affinities than calcium. The apparent disassociation constants for lanthanide ions ranged from 20 to 75 μM, which are an order of magnitude higher than the affinity for calcium, with a higher selectivity toward heavy REEs over light REEs. Most proteins release bound ions at mildly acidic conditions (pH 5-6), and the high affinity REE-binding lanmodulin protein can bind 3-4 REE ions at pH as low as ∼2.5. Circular dichroism (CD) spectra of the RTX domain demonstrate pH-induced folding of the beta roll domain in the absence of ions, indicating that protonation of key amino acids enables structure formation in low pH solutions. The beta roll domain coordinates up to four ions in extreme pH conditions (pH < 1), as determined by equilibrium ultrafiltration experiments. Finally, to demonstrate a potential application of the RTX domain, REE ions (Nd3+ and Dy3+) were recovered from other non-REEs (Fe2+ and Co2+) in a NdFeB magnet simulant solution (at pH 6).

Investigation of a Large Kindred Reveals Cardiac Calsequestrin (CASQ2) as a Cause of Brugada Syndrome.

Brugada syndrome (BrS) is an inherited primary channelopathy syndrome associated with the risk of ventricular fibrillation (VF) and sudden cardiac death in a structurally normal heart. The aim of this study was to clinically and genetically evaluate a large family with severe autosomal dominant Brugada syndrome. Clinical and genetic studies were performed. Genetic analysis was conducted with NGS technologies (WES) using the Illumina instrument. According to the standard procedure, variants found by WES were confirmed in all available families by Sanger sequencing. The effect of the variants was studied by using in silico prediction of pathogenicity. The proband was a 52-year-old man who was admitted to the emergency department for syncope at rest. WES of the index case identified a heterozygous VUS CASQ2, c.532T>C, p.(Tyr178His). We studied the segregation of the variation in all pedigree members. All the patients were heterozygous for the variation CASQ2 p.(Tyr178His), whereas the remaining healthy individuals in the family were homozygous for the normal allele. Structural analysis of CASQ2 p.(Tyr178His) was performed and revealed an important effect of the missense variation on monomer stability. The CASQ2 Tyr180 residue is located inside the sarcoplasmic reticulum (SR) junctional face membrane interaction domain and is predicted to disrupt filamentation. Our data suggest that the p.Tyr178His substitution is associated with BrS in the family investigated, affecting the stability of the protein, disrupting filamentation at the interdimer interface, and affecting the subsequent formation of tetramers and polymers that contain calcium-binding sites.

A New Perspective on the Biomineralization of Radicular Dentin

The biomineralization of radicular dentin involves complex molecular signaling. The exact mechanism of action remains elusive. However, the literature showed that protein binding sites for calcium ions and mineral precipitation are essential to promote the remineralization process. Dentin organic matrix possesses non-collagenous proteins, such as dentin matrix protein 1 (DMP1), that can initiate and regulate crystal nucleation. Recombinant DMP1 molecules have been thought to perform specific molecular recognition with the apatite surface, guiding calcium phosphate clusters during recruitment through the type I collagen matrix, which serves as a template for controlled biomineralization. The biological basis for the clinical benefits of this process has yet to be completely elucidated. From a clinical perspective, caries, acid erosion, or disinfection protocol treatments can cause dentin demineralization. Given this fact, dentin biomimetic remineralization strategies and treatment strategies aimed at preserving the dentin matrix have been proposed.

Preparation of Calcium-Binding Peptides Derived from Mackerel (Scomber japonicus) Protein and Structural Characterization and Stability Analysis of Its Calcium Complexes.

The purpose of this study was to prepare mackerel peptides (MPs) with calcium-binding capacity through an enzyme method and to investigate the potential role they play in improving the bioavailability of calcium in vitro. The calcium-binding capacity, degree of hydrolysis (DH), molecular weight (MW), and charge distribution changes with the enzymolysis time of MPs were measured. The structural characterization of mackerel peptide-calcium (MP-calcium) complexes was performed using spectroscopy and morphology analysis. The results showed that the maximum calcium-binding capacity of the obtained MPs was 120.95 mg/g when alcalase was used for 3 h, with a DH of 15.45%. Moreover, with an increase in hydrolysis time, the MW of the MPs decreased, and the negative charge increased. The carboxyl and amino groups in aspartic (Asp) and glutamate (Glu) of the MPs may act as calcium-binding sites, which are further assembled into compact nanoscale spherical complexes with calcium ions through intermolecular interactions. Furthermore, even under the influence of oxalic acid, MP-calcium complexes maintained a certain solubility. This study provides a basis for developing new calcium supplements and efficiently utilizing the mackerel protein resource.

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.

Structural bases for stoichiometry-selective calcium potentiation of a neuronal nicotinic receptor.

α4β2 nicotinic acetylcholine (nACh) receptors assemble in two stoichiometric forms, one of which is potentiated by calcium. The sites of calcium binding that underpin potentiation are not known. To identify calcium binding sites, we applied cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations to each stoichiometric form of the α4β2 nACh receptor in the presence of calcium ions. To test whether the identified calcium sites are linked to potentiation, we generated mutants of anionic residues at the sites, expressed wild type and mutant receptors in clonal mammalian fibroblasts, and recorded ACh-elicited single-channel currents with or without calcium. Both cryo-EM and MD simulations show calcium bound to a site between the extracellular and transmembrane domains of each α4 subunit (ECD-TMD site). Substituting alanine for anionic residues at the ECD-TMD site abolishes stoichiometry-selective calcium potentiation, as monitored by single-channel patch clamp electrophysiology. Additionally, MD simulation reveals calcium association at subunit interfaces within the extracellular domain. Substituting alanine for anionic residues at the ECD sites reduces or abolishes stoichiometry-selective calcium potentiation. Stoichiometry-selective calcium potentiation of the α4β2 nACh receptor is achieved by calcium association with topographically distinct sites framed by anionic residues within the α4 subunit and between the α4 and β2 subunits. Stoichiometry-selective calcium potentiation could result from the greater number of calcium sites in the stoichiometric form with three rather than two α4 subunits. The results are relevant to modulation of signalling via α4β2 nACh receptors in physiological and pathophysiological conditions.

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract

Unique patterns of inheritance and selection on Y chromosomes have led to the evolution of specialized gene functions. We report CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. We demonstrate that the sperm tails of WDY mutants beat approximately half as fast as those of wild-type and that mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract. Therefore, although mature sperm are produced by WDY mutant males, and are transferred to females, those sperm fail to enter the female sperm storage organs. We report genotype-dependent and regional differences in sperm motility that appear to break the correlation between sperm tail beating and propulsion. Furthermore, we identify a significant change in hydrophobicity at a residue at a putative calcium-binding site in WDY orthologs at the split between the melanogaster and obscura species groups, when WDY first became Y-linked. This suggests that a major functional change in WDY coincided with its appearance on the Y chromosome. Finally, we show that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

Rand Protease: The Role of Calcium-Binding Site on Stability with Mutagenesis and The Effectiveness on Leather Dehairing

Bacillus subtilis produces a number of proteases, which are highly demanded in various industries, especially the thermostable one. Rand protease, originally isolated from B. subtilis, has thermostability and other remarkable properties such as organic solvent tolerance and pH stability. However, its vulnerability to instability-induced degradation has limited its applications. Because Rand protease contains two calcium ions for folding, activation, and, above all, stability, previous studies have shown that boosting the calcium-binding affinity enhances stability. Therefore, Rand protease’s susceptibility to degradation could be remedied by discovering the calcium-binding site having the greatest impact on stability for further calcium-binding affinity improvement. This was done with an in silico mutagenesis approach whereby one calcium-binding site was mutated to alanine and evaluated either the RMSD, the deviation of the mutated configuration from the original configuration using YASARA, or stability in terms of kcal/mol using HotSpot Wizard. The result found that calcium-binding sites Leu75 from YASARA and Tyr171 from HotSpot Wizard have higher influences on stability (our target). This result was also validated using Pymol, ExPASy ProtParam, and Molprobity. Additionally, Rand protease-chemical formulation dehairs leather best without additional metal ions at pH 7.0 and for 18 h. It also produced higher-quality leather with smaller pores and softer leather than chemical formulations. In contrast, hair breakage was observed in calcium treatment, which is compatible with the low dehairing activity achieved. In conclusion, Leu75 and Tyr171 are vital for calcium stabilisation and this enzyme has demonstrated its crucial efficacy in the leather dehairing industry.

Novel Calcium-Binding Peptide from Bovine Bone Collagen Hydrolysates and Its Potential Pro-Osteogenic Activity via Calcium-Sensing Receptor (CaSR).

This paper aims to explore the osteogenic activity and potential mechanism of the peptide-calcium chelate, and provides a theoretical basis for peptide-calcium chelates as functional foods to prevent or improve osteoporosis. In this research, a novel peptide (Phe-Gly-Leu, FGL) with a high calcium-binding capacity is screened from bovine bone collagen hydrolysates (CPs), calcium binding sites of which mainly included carbonyl, amino and carboxyl groups. The FGL-Ca significantly enhances the osteogenic activity of MC3T3-E1 cells (survival rate, differentiation, and mineralization). The results of calcium fluorescence labeling and molecular docking show that FGL-Ca may activate calcium-sensing receptor (CaSR), leading to an increase in intracellular calcium concentration, then enhancing osteogenic activity of MC3T3-E1 cells. Further research found that FGL-Ca significantly promotes the mRNA and protein expression levels of CaSR, transforming growth factor β (TGF-β1), TGF-β-type II receptor (TβRII), Smad2, Smad3, osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegrin (OPG), and collagen type I (COLI). Subsequently, in the signal pathway intervention experiment, the expression levels of genes and proteins related to the TGF-β1/Smad2/3 signaling pathway that are promoted by FGL-Ca are found to decrease. These results suggest that FGL-Ca may activate CaSR, increase intracellular calcium concentration, and activate TGF-β1/Smad2/3 signaling pathway, which may be one of the potential mechanisms for enhancing osteogenic activity.

A plasmid containing the human metallothionein-II gene selectively distinguishes trivalent lanthanum from several divalent heavy metal cations during monoclonal antibody-assisted agarose gel electrophoresis.

Trivalent lanthanide ions are known for their ability to interact with calcium-binding sites in various proteins. There is a need to assess the bioavailability of lanthanides and other heavy metals introduced into the body as components of implants or as contrast agents. This study aimed to develop a method to address bioavailability and/or presence of trivalent lanthanide ions by examining electrophoretic mobility in an agarose gel of a plasmid harboring the human metallothionein-II gene (hMT-II). Mobility of the plasmid was specifically altered by a monoclonal antibody raised against the zinc-binding transcription factor that controls the activity of the hMT-II gene. This study showed that the plasmid acquired a lanthanide-specific mobility pattern that allowed the presence of lanthanide ions to be readily determined in a 0.8% agarose gel. These findings suggest that this plasmid/monoclonal antibody combination under selected conditions may be useful in industrial, environmental, and biomedical settings to identify, separate, or capture lanthanide ions in complex mixtures that contain an array of metal ions.

Synergistic effects of putative Ca2+-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus

ABSTRACT In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca2+-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca2+-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca2+-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca2+-binding sites. Western blotting implied that mutations in Ca2+-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca2+-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca2+-binding sites of AfCaM, demonstrating that Ca2+-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca2+-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.

Structure and function of the EA1 surface layer of Bacillus anthracis

The Gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a deadly disease mostly affecting wildlife and livestock, as well as representing a bioterrorism threat. Its cell surface is covered by the mutually exclusive S-layers Sap and EA1, found in early and late growth phases, respectively. Here we report the nanobody-based structural characterization of EA1 and its native lattice contacts. The EA1 assembly domain consists of 6 immunoglobulin-like domains, where three calcium-binding sites structure interdomain contacts that allow monomers to adopt their assembly-competent conformation. Nanobody-induced depolymerization of EA1 S-layers results in surface defects, membrane blebbing and cell lysis under hypotonic conditions, indicating that S-layers provide additional mechanical stability to the cell wall. Taken together, we report a complete model of the EA1 S-layer and present a set of nanobodies that may have therapeutic potential against Bacillus anthracis.

Improvement of α-amino Ester Hydrolase Stability via Computational Protein Design.

Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for β-lactam synthesis reactions. Rational design of 'whole variants' was explored to rapidly improve AEH thermostability by mutating between 3-15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.