Intestinal mucin is a chaperone of multivalent copper.

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu2+ and the other for Cu1+, the intestinal mucin, MUC2, prevents copper toxicity by blocking futile redox cycling and the squandering of dietary antioxidants, while nevertheless permitting uptake of this important trace metal into cells. These findings emphasize the value of molecular structure in advancing mucosal biology, while introducing mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones.

Structural insights into the mechanism of oxidative activation of heme-free H-NOX from Vibrio cholerae.

Bacterial heme nitric oxide/oxygen (H-NOX) domains are nitric oxide (NO) or oxygen sensors. This activity is mediated through binding of the ligand to a heme cofactor. However, H-NOX from Vibrio cholerae (Vc H-NOX) can be easily purified in a heme-free state that is capable of reversibly responding to oxidation, suggesting a heme-independent function as a redox sensor. This occurs by oxidation of Cys residues at a zinc-binding site conserved in a subset of H-NOX homologs. Remarkably, zinc is not lost from the protein upon oxidation, although its ligation environment is significantly altered. Using a combination of computational and experimental approaches, we have characterized localized structural changes that accompany the formation of specific disulfide bonds between Cys residues upon oxidation. Furthermore, the larger-scale structural changes accompanying oxidation appear to mimic those changes observed upon NO binding to the heme-bound form. Thus, Vc H-NOX and its homologs may act as both redox and NO sensors by completely separate mechanisms.

Outpatient Expectant Management of Term Prelabor Rupture of Membranes: A Retrospective Cohort Study.

OBJECTIVE: To examine outcomes among women with prelabor rupture of membranes (PROM) who declined induction and chose outpatient expectant management compared with those admitted for induction. STUDY DESIGN: This is a retrospective cohort study of term women with singleton, vertex-presenting fetuses who presented with PROM between July 2016 and June 2017 and were eligible for outpatient expectant management (n = 166). The primary outcomes were time from PROM to delivery and time from admission to delivery. Maternal and neonatal outcomes were also compared between groups. Multivariable linear regressions were used to assess time differences between groups, adjusting for known maternal and pregnancy characteristics. RESULTS: Compared with admitted patients, women managed expectantly at home had significantly longer PROM to delivery intervals (median 29.2 vs. 17 hours, p < 0.001), but were more likely to deliver within 24 hours of admission (95.1 vs. 82.9%, p = 0.004). In the adjusted analysis, PROM to delivery was 7 hours longer (95% confidence interval [CI]: 3.9-10.0) and admission to delivery was 5.3 hours shorter (95% CI: 2.8-7.7) in the outpatient expectant management cohort. There were no differences in secondary outcomes. CONCLUSION: Outpatient management of term PROM is associated with longer PROM to delivery intervals, but shorter admission to delivery intervals.

Oxytocin Compared to Buccal Misoprostol for Induction of Labor after Term Prelabor Rupture of Membranes.

OBJECTIVE: This study was aimed to determine if admission-to-delivery times vary between term nulliparous women with prelabor rupture of membranes (PROM) who initially receive oxytocin compared with buccal misoprostol for labor induction. STUDY DESIGN: This is a retrospective cohort of 130 term, nulliparous women with PROM and cervical dilation of ≤2 cm who underwent induction of labor with intravenous oxytocin or buccal misoprostol. The primary outcome was time from admission to delivery. Linear regressions with log transformation were used to estimate the effect of induction agent on time to delivery. RESULTS: Women receiving oxytocin had faster admission-to-delivery times than women receiving misoprostol (16.9 vs. 19.9 hours, p = 0.013). There were no significant differences in secondary outcomes between the groups. In the adjusted model, women who received misoprostol had a 22% longer time from admission to delivery (95% CI 5.0-42.0%) compared with women receiving oxytocin. CONCLUSION: In term nulliparous patients with PROM, intravenous oxytocin is associated with faster admission-to-delivery times than buccal misoprostol.

The FeoC [4Fe-4S] Cluster Is Redox-Active and Rapidly Oxygen-Sensitive.

The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anaerobic conditions, most bacteria utilize the widely distributed ferrous iron (Fe2+) uptake (Feo) system to import metabolically-required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it is composed of a main membrane component (FeoB) essential for iron translocation, as well as two small, cytosolic proteins (FeoA and FeoC) hypothesized to function as accessories to this process. FeoC has many hypothetical functions, including that of an iron-responsive transcriptional regulator. Here, we demonstrate for the first time that Escherichia coli FeoC (EcFeoC) binds an [Fe-S] cluster. Using electronic absorption, X-ray absorption, and electron paramagnetic resonance spectroscopies, we extensively characterize the nature of this cluster. Under strictly anaerobic conditions after chemical reconstitution, we demonstrate that EcFeoC binds a redox-active [4Fe-4S]2+/+ cluster that is rapidly oxygen-sensitive and decays to a [2Fe-2S]2+ cluster (t1/2 ≈ 20 s), similar to the [Fe-S] cluster in the fumarate and nitrate reductase (FNR) transcriptional regulator. We further show that this behavior is nearly identical to the homologous K. pneumoniae FeoC, suggesting a redox-active, oxygen-sensitive [4Fe-4S]2+ cofactor is a general phenomenon of cluster-binding FeoCs. Finally, in contrast to FNR, we show that the [4Fe-4S]2+ cluster binding to FeoC is associated with modest conformational changes of the polypeptide, but not protein dimerization. We thus posit a working hypothesis in which the cluster-binding FeoCs may function as oxygen-sensitive iron sensors that fine-tune pathogenic ferrous iron acquisition.

pH-Induced Binding of the Axial Ligand in an Engineered CuA Site Favors the πu State.

CuA centers perform efficient long-range electron transfer. The electronic structure of native CuA sites can be described by a double-potential well with a dominant σu* ground state in fast equilibrium with a less populated πu ground state. Here, we report a CuA mutant in which a lysine was introduced in the axial position. This results in a highly unstable protein with a pH-dependent population of the two ground states. Deep analysis of the high-pH form of this variant shows the stabilization of the πu ground state due to direct binding of the Lys residue to the copper center that we attribute to deprotonation of this residue.

Design of a single protein that spans the entire 2-V range of physiological redox potentials.

The reduction potential (E°') is a critical parameter in determining the efficiency of most biological and chemical reactions. Biology employs three classes of metalloproteins to cover the majority of the 2-V range of physiological E°'s. An ultimate test of our understanding of E°' is to find out the minimal number of proteins and their variants that can cover this entire range and the structural features responsible for the extreme E°'. We report herein the design of the protein azurin to cover a range from +970 mV to -954 mV vs. standard hydrogen electrode (SHE) by mutating only five residues and using two metal ions. Spectroscopic methods have revealed geometric parameters important for the high E°'. The knowledge gained and the resulting water-soluble redox agents with predictable E°'s, in the same scaffold with the same surface properties, will find wide applications in chemical, biochemical, biophysical, and biotechnological fields.

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins.

Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host-pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a "switch" role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.

De novo selection of oncogenes.

All cellular proteins are derived from preexisting ones by natural selection. Because of the random nature of this process, many potentially useful protein structures never arose or were discarded during evolution. Here, we used a single round of genetic selection in mouse cells to isolate chemically simple, biologically active transmembrane proteins that do not contain any amino acid sequences from preexisting proteins. We screened a retroviral library expressing hundreds of thousands of proteins consisting of hydrophobic amino acids in random order to isolate four 29-aa proteins that induced focus formation in mouse and human fibroblasts and tumors in mice. These proteins share no amino acid sequences with known cellular or viral proteins, and the simplest of them contains only seven different amino acids. They transformed cells by forming a stable complex with the platelet-derived growth factor ß receptor transmembrane domain and causing ligand-independent receptor activation. We term this approach de novo selection and suggest that it can be used to generate structures and activities not observed in nature, create prototypes for novel research reagents and therapeutics, and provide insight into cell biology, transmembrane protein-protein interactions, and possibly virus evolution and the origin of life.

Multicopper manganese oxidase accessory proteins bind Cu and heme.

Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

Kß Valence to Core X-ray Emission Studies of Cu(I) Binding Proteins with Mixed Methionine - Histidine Coordination. Relevance to the Reactivity of the M- and H-sites of Peptidylglycine Monooxygenase.

Biological systems use copper as a redox center in many metalloproteins, where the role of the metal is to cycle between its +1 and +2 oxidation states. This chemistry requires the redox potential to be in a range that can stabilize both Cu(I) and Cu(II) states and often involves protein-derived ligand sets involving mixed histidine-methionine coordination that balance the preferences of both oxidation states. Transport proteins, on the other hand, utilize copper in the Cu(I) state and often contain sites comprised predominately of the cuprophilic residue methionine. The electronic factors that allow enzymes and transporters to balance their redox requirements are complex and are often elusive due to the dearth of spectroscopic probes of the Cu(I) state. Here we present the novel application of X-ray emission spectroscopy to copper proteins via a study of a series of mixed His-Met copper sites where the ligand set varies in a systematic way between the His3 and Met3 limits. The sites are derived from the wild-type peptidylglycine monooxygenase (PHM), two single-site variants which replicate each of its two copper sites (CuM-site and CuH-site), and the transporters CusF and CusB. Clear differences are observed in the Kß2,5 region at the Met3 and His3 limits. CusB (Met3) has a distinct peak at 8978.4 eV with a broad shoulder at 8975.6 eV, whereas CuH (His3) has two well-resolved features: a more intense feature at 8974.8 eV and a second at 8977.2 eV. The mixed coordination sphere CusF (Met2His) and the PHM CuM variant (Met1His2) have very similar spectra consisting of two features at 8975.2 and 8977.8 eV. An analysis of DFT calculated spectra indicate that the intensity of the higher energy peak near 8978 eV is mediated by mixing of ligand-based orbitals into the Cu d(10) manifold, with S from Met providing more intensity by facilitating increased Cu p-d mixing. Furthermore, reaction of WT PHM with CO (an oxygen analogue) produced the M site CO complex, which showed a unique XES spectrum that could be computationally reproduced by including interactions between Cu(I) and the CO ligand. The study suggests that the valence-to-core (VtC) region can not only serve as a probe of ligand speciation but also offer insight into the coordination geometry, in a fashion similar to XAS pre-edges, and may be sufficiently sensitive to the coordination of exogenous ligands to be useful in the study of reaction mechanisms.

Binuclear Cu(A) Formation in Biosynthetic Models of Cu(A) in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate.

Cu(A) is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N2ORs), and nitric oxide reductase (NOR). In these proteins, the Cu(A) centers facilitate efficient ET (kET > 104s⁻¹) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of Cu(A) are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu(A) maturation process are still lacking. Previous studies of the Cu(A) assembly mechanism in vitro using a biosynthetic model Cu(A) center in azurin (Cu(A)Az) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as Cu(A)' and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu(A) assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu(A) site, with a major focus on characterization of the novel intermediate Ix. We show that Cu(A) assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that Cu(A) formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80-85% population in these variants, which allowed us to employ ultraviolet-visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)(2)(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native Cu(A), these results shed light on the structural features of the important intermediates and mechanism of Cu(A) formation.

Iron-sulfur clusters are involved in post-translational arginylation.

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF.

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.

Stable Cu(II) and Cu(I) mononuclear intermediates in the assembly of the CuA center of Thermus thermophilus cytochrome oxidase.

CuA is a dinuclear mixed-valence center located in subunit 2 of the ba(3)-type cytochrome oxidase from Thermus thermophilus. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of CuA formation from inorganic ions. The formulation of the CuA center as an electron-delocalized Cu(1.5)-Cu(1.5) system implicates both Cu(II) and Cu(I) states in the metalation process. In earlier work we showed that selenomethionine (SeM) substitution of the coordinated M160 residue provided a ligand-directed probe for studying the copper coordination environment via the Se XAS signal, which was particularly useful for interrogating the Cu(I) states where other spectroscopic probes are absent. In the present study we have investigated the formation of mixed-valence CuA and its M160SeM derivative by stopped-flow UV-vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu(I) CuA has been studied by XAS alone. Our results establish the presence of previously undetected mononuclear intermediates and show important differences from the metalation reactions of purple CuA azurin. XAS spectroscopy at Cu and Se edges has allowed us to extend mechanistic inferences to formation of the di-Cu(I) state which may be more relevant to biological CuA assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems and that the dominant intermediate along the pathway to mixed-valence is a new green species with λ(max) = 460 nm. This intermediate has been isolated in a homogeneous state and shown to be a mononuclear Cu(II)-(His)(Cys)(2) species with no observable Cu(II)-(Met) interaction. Reduction with dithionite generates its Cu(I) homologue which is again mononuclear but now shows a strong interaction with the Met160 thioether. The results are discussed within the framework of the "coupled distortion" model for Cu(II) thiolates and their relevance to biological metalation reactions of the CuA center.

Efficacy and safety of four COVID-19 vaccines in preventing SARS-CoV-2 infection: A rapid review. / Eficacia y seguridad de cuatro COVID-19 vacunas para prevenir la infección por SARS-CoV-2: una revisión rápida

INTRODUCTION: Since the emergence of the SARS-CoV-2, there have been efforts to develop vaccines to control the COVID-19 pandemic. OBJECTIVE: The present study assessed the efficacy and safety of the BNT162b2, mRNA-1273, ChAdOx1/AZD1222 and Gam-COVID-Vac rAd26-S/rAd5-S vaccines against the SARS-CoV-2. MATERIALS AND METHODS: We searched PubMed/MEDLINE, Google Scholar, Cochrane, and the WHO International Clinical Trials Registry Platform on March 15, 2021. The search terms used were: "vaccine" OR "vaccination" AND "covid19" OR "coronavirus" OR "sarscov2" AND "bnt162b2" OR "chadox1-S" OR "azd1222" OR "sputnik" OR "Gam-COVID-Vac" OR "mrna" OR "mRNA-1273" . We measured the risk of bias of the studies and the quality of the evidence using GRADE profiles. A qualitative and quantitative analysis of the results of clinical trials is presented. RESULTS: Of the 74 identified studies, 4 were finally included in this review. The efficacies of the BNT162b2, mRNA-1273, ChAdOx1/AZD1222 and Gam-COVID-VacrAd26-S/rAd5-S vaccines against symptomatic COVID-19 were 95,0% (CI95% 90,3-97,6), 94,1% (CI95% 89,3-96,8), 66,7% (CI95% 57,4-74,0), and 91,1% (CI95% 83,8-95,1), respectively. There was moderate certainty of the evidence due to serious indirectness, when we measured the risk of bias of the studies and the quality of the evidence using GRADE profile. The safety profiles were acceptable, and data on serious adverse events (summary RR=0,93; CI95% 0,77-1,12; p=0,16) and deaths from all causes (summary RR=0,70; CI95% 0,33-1,50; p=0,90) showed no significant differences. CONCLUSION: The results of this review support the level of evidence for the efficacy and safety of the COVID-19 vaccines analysed.

Introducción. Desde que surgió el virus SARS-CoV-2, se han realizado esfuerzos para desarrollar vacunas para controlar la pandemia por COVID-19. Objetivo. Evaluar los datos de la eficacia y seguridad de las vacunas BNT162b2, mRNA-1273, ChAdOx1/AZD1222 y Gam-COVID-Vac rAd26-S/rAd5-S contra el SARS-CoV-2. Materiales y métodos. Se realizaron búsquedas en PubMed/MEDLINE, Google Scholar, Cochrane y la Plataforma de Registro Internacional de Ensayos Clínicos de la OMS el 15 de marzo de 2021. Los términos usados fueron: "vaccine" OR "vaccination" AND "covid19" OR "coronavirus" OR "sarscov2" AND "bnt162b2" OR "chadox1-S" OR "azd1222" OR "sputnik" OR "Gam-COVID-Vac" OR "mrna" OR "mRNA-1273". Se midió el riesgo de sesgo de los estudios y la calidad de la información por medio de los perfiles GRADE. Se presenta un análisis cualitativo y cuantitativo de los resultados de los estudios clínicos. Resultados. Se identificaron 74 estudios y se incluyeron 4 en la revisión. La eficacia de las vacunas BNT162b2, mRNA-1273, ChAdOx1/AZD1222 y Gam-COVID-VacrAd26-S/rAd5-S contra la COVID-19 sintomática fue del 95,0 % (IC95% 90,3-97,6), 94,1 % (IC95% 89,3-96,8), 66,7 % (IC95% 57,4-74,0) y 91,1 % (IC95% 83,8-95,1), respectivamente, y hubo una certeza moderada de la información debido a la falta de evidencia directa. Los perfiles de seguridad fueron aceptables, y los eventos adversos graves (RR resumido=0,93; IC95% 0,77-1,12; p=0,16) y muerte por todas las causas (RR resumido=0,70; IC95% 0,33-1,50; p=0,90) no mostraron diferencias significativas. Conclusión. Los resultados de esta revisión respaldan el nivel de evidencia de la eficacia y seguridad de las vacunas COVID-19 que fueron analizadas.

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins.

Zinc acquisition from limited environments is critical for bacterial survival and pathogenesis. AztD has been identified as a periplasmic or cell surface zinc-binding protein in numerous bacterial species. In Paracoccus denitrificans, AztD can transfer zinc directly to AztC, the solute binding protein for a zinc-specific ATP-binding cassette transporter system, suggesting a role in zinc acquisition and homeostasis. Here, we present the first cry stal structures of AztD from P. denitrificans and tbe human pathogen Citrobacter koseri, revealing a beta-propeller fold and two high-affinity zinc-binding sites that are highly conserved among AztD homologs. These structures combined with transfer assays using WT and mutant proteins provide rare insight into the mechanism of direct zinc transfer from one protein to another. Given the importance of zinc import to bacterial pathogenesis, these insights may prove valuable to the development of zinc transfer inhibitors as antibiotics.

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli.

Escherichia coli CusCBAF represents an important class of bacterial efflux pump exhibiting selectivity towards Cu(I) and Ag(I). The complex is comprised of three proteins: the CusA transmembrane pump, the CusB soluble adaptor protein, and the CusC outer-membrane pore, and additionally requires the periplasmic metallochaperone CusF. Here we used spectroscopic and kinetic tools to probe the mechanism of copper transfer between CusF and CusB using selenomethionine labeling of the metal-binding Met residues coupled to RFQ-XAS at the Se and Cu edges. The results indicate fast formation of a protein-protein complex followed by slower intra-complex metal transfer. An intermediate coordinated by ligands from each protein forms in 100 ms. Stopped-flow fluorescence of the capping CusF-W44 tryptophan that is quenched by metal transfer also supports this mechanism. The rate constants validate a process in which shared-ligand complex formation assists protein association, providing a driving force that raises the rate into the diffusion-limited regime.

Effect of circular permutation on the structure and function of type 1 blue copper center in azurin.

Type 1 copper (T1Cu) proteins are electron transfer (ET) proteins involved in many important biological processes. While the effects of changing primary and secondary coordination spheres in the T1Cu ET function have been extensively studied, few report has explored the effect of the overall protein structural perturbation on active site configuration or reduction potential of the protein, even though the protein scaffold has been proposed to play a critical role in enforcing the entatic or "rack-induced" state for ET functions. We herein report circular permutation of azurin by linking the N- and C-termini and creating new termini in the loops between 1st and 2nd ß strands or between 3rd and 4th ß strands. Characterization by electronic absorption, electron paramagnetic spectroscopies, as well as crystallography and cyclic voltammetry revealed that, while the overall structure and the primary coordination sphere of the circular permutated azurins remain the same as those of native azurin, their reduction potentials increased by 18 and 124 mV over that of WTAz. Such increases in reduction potentials can be attributed to subtle differences in the hydrogen-bonding network in secondary coordination sphere around the T1Cu center.

Efficacy and safety of four COVID-19 vaccines in preventing SARS-CoV-2 infection: A rapid review / Eficacia y seguridad de cuatro vacunas COVID-19 para prevenir la infección por SARS-CoV-2: una revisión rápida

Introduction: Since the emergence of the SARS-CoV-2, there have been efforts to develop vaccines to control the COVID-19 pandemic. Objective: The present study assessed the efficacy and safety of the BNT162b2, mRNA-1273, ChAdOx1/AZD1222 and Gam-COVID-Vac rAd26-S/rAd5-S vaccines against the SARS-CoV-2. Materials and methods: We searched PubMed/MEDLINE, Google Scholar, Cochrane, and the WHO International Clinical Trials Registry Platform on March 15, 2021. The search terms used were: "vaccine" OR "vaccination" AND "covid19" OR "coronavirus" OR "sarscov2" AND "bnt162b2" OR "chadox1-S" OR "azd1222" OR "sputnik" OR "Gam-COVID-Vac" OR "mrna" OR "mRNA-1273" . We measured the risk of bias of the studies and the quality of the evidence using GRADE profiles. A qualitative and quantitative analysis of the results of clinical trials is presented. Results: Of the 74 identified studies, 4 were finally included in this review. The efficacies of the BNT162b2, mRNA-1273, ChAdOx1/AZD1222 and Gam-COVID-VacrAd26-S/rAd5-S vaccines against symptomatic COVID-19 were 95,0% (CI95% 90,3-97,6), 94,1% (CI95% 89,3-96,8), 66,7% (CI95% 57,4-74,0), and 91,1% (CI95% 83,8-95,1), respectively. There was moderate certainty of the evidence due to serious indirectness, when we measured the risk of bias of the studies and the quality of the evidence using GRADE profile. The safety profiles were acceptable, and data on serious adverse events (summary RR=0,93; CI95% 0,77-1,12; p=0,16) and deaths from all causes (summary RR=0,70; CI95% 0,33-1,50; p=0°90) showed no significant differences. Conclusion: The results of this review support the level of evidence for the efficacy and safety of the COVID-19 vaccines analysed.

Introducción. Desde que surgió el virus SARS-CoV-2, se han realizado esfuerzos para desarrollar vacunas para controlar la pandemia por COVID-19. Objetivo. Evaluar los datos de la eficacia y seguridad de las vacunas BNT162b2, mRNA-1273, ChAdOx1/AZD1222 y Gam-COVID-Vac rAd26-S/rAd5-S contra el SARS-CoV-2. Materiales y métodos. Se realizaron búsquedas en PubMed/MEDLINE, Google Scholar, Cochrane y la Plataforma de Registro Internacional de Ensayos Clínicos de la OMS el 15 de marzo de 2021. Los términos usados fueron: "vaccine" OR "vaccination" AND "covid19" OR "coronavirus" OR "sarscov2" AND "bnt162b2" OR "chadox1-S" OR "azd1222" OR "sputnik" OR "Gam-COVID-Vac" OR "mrna" OR "mRNA-1273'.' Se midió el riesgo de sesgo de los estudios y la calidad de la información por medio de los perfiles GRADE. Se presenta un análisis cualitativo y cuantitativo de los resultados de los estudios clínicos. Resultados. Se identificaron 74 estudios y se incluyeron 4 en la revisión. La eficacia de las vacunas BNT162b2, mRNA-1273, ChAdOx1/AZD1222 y Gam-COVID-VacrAd26-S/rAd5-S contra la COVID-19 sintomática fue del 95,0 % (IC95% 90,3-97,6), 94,1 % (IC95% 89,3-96,8), 66,7 % (IC95% 57,4-74,0) y 91,1 % (IC95% 83,8-95,1), respectivamente, y hubo una certeza moderada de la información debido a la falta de evidencia directa. Los perfiles de seguridad fueron aceptables, y los eventos adversos graves (RR resumido=0,93; IC95% 0,77-1,12; p=0,16) y muerte por todas las causas (RR resumido=0,70; IC95% 0,33-1,50; p=0,90) no mostraron diferencias significativas. Conclusión. Los resultados de esta revisión respaldan el nivel de evidencia de la eficacia y seguridad de las vacunas COVID-19 que fueron analizadas.