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Abstract: Extracellular heat shock proteins have been reported to participate in both innate and adaptive immune responses. We have found that recombinant mouse inducible heat shock protein 72 (Hsp72) bound to lymphoid neoplastic P388D1 cells. In the present study, we examined which region of mouse Hsp72 interacted with this cell line by using truncated variants that are sequentially lacking sections of the C-terminal region. The full-length mouse Hsp72 specifically bound to P388D1 cells, but not to mastocytoma P815 cells. Deletion of the C-terminal tail portion of mouse Hsp72 markedly decreased the binding to P388D1 cells and the sequential truncation of the C-terminal helical region led to a loss of binding activity. Specific binding was not observed for either the variant with a minimal substrate-binding structure or the ATP-binding domain alone. On the other hand, two truncated variants lacking the ATP-binding domain significantly bound to P388D1 cells. However, the variant lacking the substrate-binding domain did not show any binding to this cell line. These results suggest that the activity to bind P388D1 cells is attributable to the C-terminal region of mouse Hsp72 in combination with the substrate-binding domain. Interestingly, the binding of mouse Hsp72 to P388D1 cells was competed by the variant with the C-terminal flexible tail sequence, but not by the variant without that sequence. These competitive experiments imply that there may be at least two membrane receptors on P388D1 cells and also that both receptors may recognize the various structures in the C-terminal region of the Hsp70 family for regulation of innate immunity. [Copyright &y& Elsevier]

Temperature transduction in mammals is possible because of the presence of a set of temperature-dependent transient receptor potential (TRP) channels in dorsal root ganglia neurons and skin cells. Six thermo-TRP channels, all characterized by their unusually high temperature sensitivity (Q10 > 10), have been cloned: TRPV1-4 are heat activated, whereas TRPM8 and TRPA1 are activated by cold. Because of the lack of structural information, the molecular basis for regulation by temperature remains unknown. In this study, we assessed the role of the C-terminal domain of thermo-TRPs and its involvement in thermal activation by using chimeras between the heat receptor TRPV1 and the cold receptor TRPM8, in which the entire C-terminal domain was switched. Here, we demonstrate that the C-terminal domain is modular and confers the channel phenotype regarding temperature sensitivity, channel gating kinetics, and PIP2 (phosphatidylinositol-4,5-bisphophate) modulation. Thus, thermo-TRP channels contain an interchangeable specific region, different from the voltage sensor, which allows them to sense temperature stimuli. [ABSTRACT FROM AUTHOR]

E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data. Recently, we described the expression and purification of E6-C 4C/4S, a stable, folded mutant of the C-terminal domain of HPV16 E6. Here, we have produced15N-labelled samples of E6-C 4C/4S for structural studies by NMR. We have assigned most1H and15N resonances and identified the elements of secondary structure of the domain. The domain displays an original a/ß topology with roughly equal proportions of a-helix and ß-sheet. The PDZ-binding region of E6, located at the extreme C-terminus of the domain, is in a random conformation. Mass spectrometry demonstrated the presence of one zinc ion per protein molecule. Kinetics of replacement of zinc by cadmium followed by1H,15N-HSQC experiments revealed specific frequency changes for the zinc-binding cysteines and their immediate neighbours. NMR spectra were affected by severe line-broadening effects which seriously hindered the assignment work. Investigation of these effects by15N relaxation experiments showed that they are due to heterogeneous dynamic behaviour with µs-ms time scale motions occurring in localised regions of the monomeric domain. [ABSTRACT FROM AUTHOR]

The process of eukaryotic translation initiation can be regulated by a highly conserved mechanism involving the phosphorylation of the translation initiation factor eIF2 on the α subunit. This mechanism is recognized as an efficient step in the host antiviral response. Vaccinia virus (VV), like many other viruses, encodes proteins to overcome this inhibitory process. The C-terminus of the vaccinia virus E3L is known to bind to double-stranded RNA (dsRNA) thereby sequestering the activator of this antiviral response. In this report, the N-terminus of E3L was found to be required for the additional regulation of eIF2α phosphorylation. This phosphorylation event did not lead to a global shutdown in protein synthesis. Because the N-terminus of E3L is required for full viral pathogenesis in mice, these results suggest an alternative role of eIF2α phosphorylation in regulating viral replication. [Copyright &y& Elsevier]

RNA polymerase II (pol II) transcribes the most varied group of genes and is present in hypo- and hyperphosphorylated forms, with residues Ser2 and Ser5 of the C-terminal domain (CTD) of the largest subunit as main targets of phosphorylation. The elongating (active) form is phosphorylated on Ser2 and can be specifically recognized with the H5 antibody. It has been found in different nuclear distributions: in discrete sites throughout the nucleoplasm, consistent with a role in transcription, and/or concentrated in “splicing speckles”, a nuclear compartment mostly devoid of transcriptional activity. Here, we assess the effects of cell fixation and permeabilization on the distribution of polymerase II and correlate its distribution with the preservation of cellular ultrastructure. We show that phospho-Ser2 polymerase II can redistribute to, or be differentially retained in, “speckles” in conditions that do not preserve cellular ultrastructure. The fixation protocols that disrupt polymerase II distribution also cause partial or total loss of TATA-binding protein, Sm antigen and PML staining in PML bodies, and have no noticeable effect in the labeling of SC35 in “splicing speckles” or coilin in Cajal bodies. When nuclear ultrastructure is preserved, phospho-Ser2 polymerase II is found in discrete sites throughout the nucleoplasm, without visible enrichment within splicing speckles. A minor proportion of the total amount of the phospho-Ser2 form is present in these domains. [Copyright &y& Elsevier]

α-Amylases are present in all kingdoms of the living world. Despite strong conservation of the tertiary structure, only a few amino acids are conserved in interkingdom comparisons. Animal α-amylases are characterized by several typical motifs and biochemical properties. A few cases of such α-amylases have been previously reported in some eubacterial species. We screened the bacterial genomes available in the sequence databases for new occurrences of animal-like α-amylases. Three novel cases were found, which belong to unrelated bacterial phyla: Chloroflexus aurantiacus, Microbulbifer degradans, and Thermobifida fusca. All the animal-like α-amylases in Bacteria probably result from repeated horizontal gene transfer from animals. The M. degradans genome also contains bacterial-type and plant-type α-amylases in addition to the animal-type one. Thus, this species exhibits α-amylases of animal, plant, and bacterial origins. Moreover, the similarities in the extra C-terminal domains (different from both the α-amylase domain C and the starch-binding domain), when present, also suggest interkingdom as well as intragenomic shuffling. [ABSTRACT FROM AUTHOR]

Human apolipoprotein E (apoE) is composed of an N-terminal (NT) domain (residues 1–191) that bears low-density lipoprotein receptor-binding sites, and a C-terminal (CT) domain (residues 210–299), which houses lipoprotein binding and apoE self-association sites. The NT domain is comprised of a four-helix bundle, while the structural organization of the CT domain is not known. Secondary structural algorithms predict that the apoE CT domain adopts an amphipathic α-helical conformation. On the basis of further sequence predictions, we identified a segment (residues 218–266) in the apoE CT domain that bears a high propensity to form a coiled-coil helix, which coincides with the putative lipoprotein-binding surface. An apoE construct bearing residues 201–299 that encompasses the entire CT domain was designed, expressed in Escherichia coli and purified by affinity chromatography. Circular dichroism (CD) spectroscopy of the apoE CT domain reveals spectra characteristic of coiled-coil helices, with the ratio of molar ellipticities at 222 nm and 208 nm ([θ]222/[θ]208) of 1.03. Trifluoroethanol (TFE) stabilized the secondary structure of the apoE CT domain and disrupted coiled-coil helix formation as determined by CD and tryptophan fluorescence analysis. Analytical ultracentrifugation and lysine-specific cross-linking analysis of the apoE CT domain revealed predominant formation of dimeric and tetrameric species in aqueous buffers, and monomeric forms in 50% TFE. Guanidine hydrochloride-induced denaturation studies reveal that, at low concentrations of denaturant, the apoE CT domain maintains the [θ]222/[θ]208 ratio at ∼1.0 and elicits an altered tertiary environment with a shift in oligomeric state towards a dimer, indicative of the role of coiled-coil helix formation in inter molecular interactions. Further, coiled-coil formation is disrupted by protonation below pH 6.0, with a corresponding decrease in Trp fluorescence emission intensity, demonstrating that salt-bridge interactions play a critical role in maintaining the structural integrity of the apoE CT domain. The data support the concept that inter molecular coiled-coil helix formation is an essential structural feature of the apoE CT domain, which likely plays a role in clustering heparin-binding sites and/or sequestering the lipid-binding surface in lipid-free states. [Copyright &y& Elsevier]

We propose three-dimensional models corresponding to the C-terminal domain of human αA- and αB-crystallins by using the comparative modeling program Modeler and the more closely related crystal structure of the small heat-shock protein (sHSP) belonging to the eukaryotic species from wheat HSP16.9 as template structure. The sequence alignments differ slightly from alignments that were used previously to construct alpha-crystallin models based on homology and the crystal structure of the more distantly related small heat-shock protein from archaeal species; Methanococcus jannaschii Mj HSP16.5, the only related structure then available as a template. The alpha-crystallin models based on HSP16.9 show better 3-D profile scores and reflect the relative shifts in the β-strands corresponding to the β-sandwich associated with the core C-terminal domain that is common to small heat-shock proteins and the alpha-crystallins. The loop between the equivalent β5–β7 strands corresponds to a region of seven amino acid residues deletion in alpha-crystallins and defines the new set of amino acid residues likely to be associated with a dimer interface. The models may be useful to examine sites of mutations that are known to affect chaperone-like activity and provide the structural basis for dimerization in alpha-crystallins. [Copyright &y& Elsevier]

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm establishing novel interactions between host and parasite proteins, particularly at the membrane skeleton that modifies both the structural and functional properties of the red cell. We present evidences that two members of the P. falciparum acyl-CoA synthetase (PfACS) family, responsible for the activation of long-chain fatty acids by thio-esterification with CoA, are transported in vesicle-like structures toward the host erythrocyte cytoplasm where they interact with the cytoskeletal protein ankyrin. Carboxyl-terminal domain (CTD) overlay studies indicated that PfACS1 and PfACS3 bind to the 78-kDa fragment of ankyrin corresponding with its spectrin-binding domain. Co-immunoprecipitation of ankyrin and PfACS1/3 indicates that at least a fraction of these proteins are physically associated in the infected erythrocytes and provide evidence for a novel specific interaction which suggest that such a binding may bring these enzymes closer to the host erythrocyte membrane where exogenous fatty acids are available. [Copyright &y& Elsevier]

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzainΔc) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography. [Copyright &y& Elsevier]

The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2′ subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH–activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis–Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589–1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E-1, which are significantly higher for cruzipain. The corresponding ΔS1 and ΔS-1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain. [ABSTRACT FROM AUTHOR]

The outbreak of COVID-19 caused by SARS-CoV-2 virus continually led to infect a large population worldwide. Currently, there is no specific viral protein-targeted therapeutics. The Nucleocapsid (N) protein of the SARS-CoV-2 virus is necessary for viral RNA replication and transcription. The C-terminal domain of N protein (CTD) involves in the self-assembly of N protein into a filament that is packaged into new virions. In this study, the CTD (PDB ID: 6WJI) was targeted for the identification of possible inhibitors of oligomerization of N protein. Herein, multiple computational approaches were employed to explore the potential mechanisms of binding and inhibitor activity of five antiviral drugs toward CTD. The five anti-N drugs studied in this work are 4E1RCat, Silmitasertib, TMCB, Sapanisertib, and Rapamycin. Among the five drugs, 4E1RCat displayed highest binding affinity (-10.95 kcal/mol), followed by rapamycin (-8.91 kcal/mol), silmitasertib (-7.89 kcal/mol), TMCB (-7.05 kcal/mol), and sapanisertib (-6.14 kcal/mol). Subsequently, stability and dynamics of the protein-drug complex were examined with molecular dynamics (MD) simulations. Overall, drug binding increases the stability of the complex with maximum stability observed in the case of 4E1RCat. The CTD-drug complex systems behave differently in terms of the free energy landscape and showed differences in population distribution. Overall, the MD simulation parameters like RMSD, RMSF, Rg, hydrogen bonds analysis, PCA, FEL, and DCCM analysis indicated that 4E1RCat and TMCB complexes were more stable as compared to silmitasertib and sapanisertib and thus could act as effective drug compounds against CTD.Communicated by Ramaswamy H. Sarma.

During lytic infection, herpes simplex virus (HSV) 1 induces a rapid shutoff of host RNA synthesis while redirecting transcriptional machinery to viral genes. In addition to being a major human pathogen, there is burgeoning clinical interest in HSV as a vector in gene delivery and oncolytic therapies, necessitating research into transcriptional control. This review summarizes the array of impacts that HSV has on RNA Polymerase (Pol) II, which transcribes all mRNA in infected cells. We discuss alterations in Pol II holoenzymes, post-translational modifications, and how viral proteins regulate specific activities such as promoter-proximal pausing, splicing, histone repositioning, and termination with respect to host genes. Recent technological innovations that have reshaped our understanding of previous observations are summarized in detail, along with specific research directions and technical considerations for future studies. [ABSTRACT FROM AUTHOR]

In a previous report, we documented that a major portion of the nuclear protein kinase CK2α (CK2α) subunit does not form heterooligomeric structures with the β subunit, but it binds tightly to nuclear structures in an epithelial Chironomus cell line [1]. We report here that the CK2α, but not β, subunit is co-localized with productively transcribing RNA polymerase II (pol II) on polytene chromosomes of Chironomus salivary gland cells. Likewise, the RAP74 subunit of TFIIF, a potential substrate for CK2, is co-localized with pol II. The occupancies of chromosomes with the CK2α and RAP74 subunits are sensitive to DRB, an inhibitor of pol II-based transcription and the activity of CK2 and pol II carboxyl-terminal kinases. DRB alters the chromosomal distribution of the CK2α and RAP74 subunits: there is a time-dependent clearance from the chromosomes of CK2α and RAP74 subunits, which coincides in time the completion and release of preinitiated transcripts after addition of DRB. The results suggest that both the CK2α and RAP74 subunits travel with the elongating pol II molecules along the DNA template during the entire transcription cycle. No detectable re-association of CK2α and RAP74 with the promoters takes, however, place after the completion of the preinitiated transcripts in the presence of DRB. In contrast, the binding of hypophosporylated pol II and TFIIH to the active gene loci is not abolished by the DRB regimen. Our data are consistent with the possibility that in living Chironomus salivary gland cells, DRB interferes with the recruitment of TFIIF, but not of TFIIH, to the promoter by interference with the activity of the CK2α subunit enzyme and phosphorylation of RAP74 and thereby DRB blocks transcription initiation. [ABSTRACT FROM AUTHOR]

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF-hand calcium-binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C-terminus of α-spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0,1–1,0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N-terminal and C-terminal halves which fold independently. Only the N-terminal subdomain binds calcium. These data suggest that the C-terminus of α-spectrin has a domain with a calmodulin fold and two calcium-binding sites. Sequence alignments suggest that the related domains in α-actinin, and possibly in dystrophin, may share the same calmodulin-like structure. However, only non-muscle α-actinins appear to have one or two EF-hand(s) with the calcium-binding consensus sequence, and a strict consensus is not found in the muscle α-actinins or dystrophins. [ABSTRACT FROM AUTHOR]

Novel mutations in the thiazide-sensitive NaCl cotransporter gene in patients with Gitelman syndrome. Gitelman syndrome (familial hypokalemia-hypomagnesemia syndrome) is an autosomal recessive inherited renal disorder characterized by defective tubular reabsorption of magnesium and potassium. In this study a group of 18 unrelated and 2 related Gitelman patients, collected from six different countries have been screened for mutations in the human thiazide-sensitive sodium-chloride cotransporter (SLC12A3) gene. Fourteen novel SLC12A3 mutations are presented along with six mutations described earlier, and three neutral polymorphisms. Among the tested patients are two who carry a total of three heterozygous SLC12A3 mutations. Two-thirds of the total number of mutant SLC12A3 alleles are amino acid substitutions. Most SLC12A3 gene mutations, 14 out of a total of 20, are localized at the intracellular carboxy-terminal domain of the NCCT protein. The pathogenicity of individual SLC12A3 mutations is based upon their predicted effect on SLC12A3 protein, and segregation in family members. Evolutionary conservation of substituted amino acid residues and their frequency in control chromosomes is presented. Identical mutations have been found in Gitelman families from different geographical origin, suggesting ancient mutations originating from a common ancestor. As yet, we have not found any evidence for a possible genotype-phenotype correlation. [ABSTRACT FROM AUTHOR]

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751–15759], Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both α and β subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the α and β subunits of αβ tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl liter or the exposure of hydrophobic areas of this subtilisin-treated tubulin (αsβs). Moreover, IKP104 lowers the sulfhydryl titer of αsβs tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of αsβs tubulin. The cleavage at the C-terminal ends of both α and β modulates the effect of IKP104 on the β subunit, but not on the α subunit. Fluorometric binding data analysis suggests that IKP104 binds to the αsβs tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for α and β and the fluoromelric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of α and β and the effect of the high-affinity site is restricted largely to the α subunit, while the low-affinity-site binding is modulated by the C-terminal domain of β. It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively. [ABSTRACT FROM AUTHOR]

Tubulin, the major component of microtubules, has a tendency to lose its ability to assemble or to bind to ligands in a time-dependent process known as “decay.” The decay process also causes tubulin to expose sulfhydryl groups and hydrophobic areas. The antimitotic drug phomopsin A strongly protects the tubulin molecule from decay. Here we have studied the interaction of phomopsin A with αβ tubulin and tubulin which has been treated with subtilisin to remove selectively the C-termini of the α and β chains (αsβs). The binding of phomopsin A to αβ tubulin decreases the sulfhydryl titer by approximately 1.0 mol/mol. Selective removal of the peptides from the C-terminal ends does not affect phomopsin A's interaction with tubulin. Moreover, the αsβs tubulin–phomopsin A complex appears to be more stable than the αβ tubulin–phomopsin A complex as determined by the time-dependent increase in exposure of sulfhydryl groups and hydrophobic areas on tubulin. In fact, phomopsin A inhibits the decay process of αsβs tubulin completely. This observation raises the possibility of determining the conformtion of this configuration of tubulin. [ABSTRACT FROM AUTHOR]

The article details a study conducted in India which amplified and characterized retinoic acid inducible gene-I (RIG-I) in goose using conventional reverse transcription method and sequencing, respectively. It mentioned the phylogeny of RIG-I which showed that it is conserved throughout vertebrate but it is apparently not present in chicken. It also discusses the closeness of chicken RIG-I like receptors with goose RIG-I and the polymorphism of C-terminal domain of RIG-I gene.

Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III-IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease., (© 2021 The Author(s).)

The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters. [ABSTRACT FROM AUTHOR]

The C-terminal domain (CTD) of MMP-2, which includes a hemopexin-like domain, has been increasingly studied as an alternative target in developing selective intervention strategies towards MMP-2. Moreover, The CTD itself has been implicated in a growing number of biological events, either MMP-dependent or –independent. The production of CTD, however, has been mostly based on the uncontrolled lysis of the latent ProMMP-2 or fusion protein expression that leaves a fusion tag. In this work we present a facile production of the untagged CTD in E. coli. The target protein was expressed as inclusion bodies, and we established an efficient wash and refolding strategy that allows us to obtain the target protein in extremely high purity. The yield was established at ~6 mg/L of the culture medium, which would greatly facilitate the production and hence the biological study of CTD. The method described herein might also prove useful for related (domain) proteins in MMP family and beyond. • Facile expression and purification of the untagged C-terminal domain of MMP-2. • Washing steps and a pH 10 refolding buffer lead to correct folding with high purity. • The refolded protein is active in binding to its physiological inhibitor—TIMP-2. [ABSTRACT FROM AUTHOR]

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca 2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca 2+ - and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca 2+ -independent basal activity and Ca 2+ -dependent activation of [ 3 H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca 2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca 2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations., Competing Interests: Conflict of interest W. G. is a recipient of the Alberta Innovates-Health Solutions Graduate Studentship Award; J. W. is a recipient of the Libin Cardiovascular Institute and Cumming School of Medicine Postdoctoral Fellowship Award; B. S. is a recipient of the Heart and Stroke Foundation of Canada Junior Fellowship Award and the Alberta Innovates-Health Solutions Fellowship Award; and S. R. W. C. is the Heart and Stroke Foundation Chair in cardiovascular research. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4 kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20 Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% ( VM = 2.16 Å3 Da−1). [ABSTRACT FROM AUTHOR]

HIV-1 capsid protein (CA) encloses the viral RNA genome and forms a conical-shaped particle in the mature HIV-1 virion, with orderly capsid assembly and disassembly critically important for viral infectivity. The 231 residue CA is composed of two helical domains, connected by a short linker sequence. In solution, CA exhibits concentration dependent dimerization which is mediated by the C-terminal domain (CTD). Here, we present nearly complete 1H, 15N and 13C assignments for the 20 kDa homodimeric CA–CTD, a prerequisite for structural characterization of the CA–CTD dimer. [ABSTRACT FROM AUTHOR]