Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis

The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in humanDPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems

Structural dynamics of Neuroglobin: photoreduction and phodissociation intermediates

Neuroglobin (Ngb) is a member of the globin family expressed in the vertebrate brain and retina; it is involved in brain protection, especially after ischemia, but its mechanism of action remains elusive. The structural characterization of Ngb in its unbound and CO bound states has revealed the presence of a very large hydrophobic cavity and a wide heme sliding to allow gaseous molecule binding (1,2). These features are most likely connected with the activity of Ngb, that could consist of radical scavenging, namely NO, relying on the presence internal docking sites to enhance availability of gaseous ligands at the active site (3,4), during pathological or physiological conditions. We have been interested to provide a description of ligand pathways within the protein matrix and their role in its function. We have investigated the Ngb protein matrix with different tecniques: UV-Vis Microspectrophotometry on crystal (off-line and on-line), X-Ray Diffraction both at low and room and X-ray Absorption Spectroscopy in solution and on a single crystal. From our data we have found gas molecule (CO photolized and dioxygen)in three different sites and we hypothesize a possible distal ligand pathway toward or from the heme-ironduring which the following sites are occuoied: i) the XeIII binding site ii) the water 9 site and the iii) distal site of the heme, from which eventually binding is achieved.References 1. Vallone B, Nienhaus K, Brunori M, Nienhaus G.U. (2004) Proteins. 56, 85-92. 2. Vallone, B., Nienhaus K., Matthes A., Brunori M., and Nienhaus G.U. (2004b) Proc. Natl. Acad. Sci. USA, 101,17351-17356. 3. Brunori M., Giuffrè A., Nienhaus K., Nienhaus G.U., Scandurra F.M., Vallone B. (2005) Proc. Natl Acad Sci U S A. 102,8483-8. 4. Anselmi M., Brunori M., Vallone B., Di Nola A. (2007) Biophys J. 93, 434-41. 5. Moschetti T., Mueller U., Schulze J., Brunori M., Vallone B. (2009) Biophys. J., 97, 1700-1708

An open flow helium cryostat for synchrotron X-ray diffraction experiments

Open flow Helium cryostats, directly blowing a stream of cold gas over the sample, remain an attractive alternative to closed cryostats in a number of cases despite the high Helium cost and often unstable and difficult operation including the growth of ice crystals on the sample: they offer open access to the sample which may be rotated over large angles allowing for large diffraction angles and simultaneous visible optical access for spectroscopy techniques. We have designed and built a new open flow Helium cryostat, making use of the paraphernalia of a commercial cryostat as much as possible. The cryostat has a temperature range from 4.5 up to 100 Kelvin for a liquid Helium consumption of around 2 l/hr when cycled between 5 K and 50 K at constant flow. The use of the new cryostat allows data collection and structure determination below 10 K; at these temperatures it is possible to trap reaction intermediates in proteins in a frozen state, previously only identified spectroscopically. We have successfully carried out X-ray diffraction data collection of murine neuroglobin at 10 K

Redirecting P450 EryK specificity by rational site-directed mutagenesis

The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such by-product decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable to industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, E89L) have been designed to allow hydroxylation of the non-physiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analysed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure and locking was recovered

Failure of apoptosis-inducing factor to act as neuroglobin reductase

International audienceNeuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, where it protects neurons against hypoxia. Ferrous Ngb has been proposed to favor cell survival by scavenging NO and/or reducing cytochrome c released into the cytosol during hypoxic stress. Both catalytic functions require an as yet unidentified Ngb-reductase activity. Such an activity was detected both in tissue homogenates of human brain and liver and in Escherichia coli extracts. Since NADH:flavorubredoxin oxidoreductase from E. coli, that was shown to reduce ferric Ngb, shares sequence similarity with the human apoptosis-inducing factor (AIF), AIF has been proposed by us as a candidate Ngb reductase. In this study, we tested this hypothesis and show that the Ngb-reductase activity of recombinant human AIF is negligible and hence incompatible with such a physiological function

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin.

International audienceThe acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7)Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed

The ALA5/ALA6/ALA7 repeat polymorphisms of the glutathione peroxidase-1 (GPx1) gene and autism spectrum disorder

: Autism is a severe neurodevelopmental disorder leading to deficits in social interaction, communication, and several activities. An increasing number of evidence suggests a role of oxidative stress in the etiology of autism spectrum disorder (ASD). Indeed, impaired antioxidant mechanisms may lead to the inadequate removal of H2 O2 with a consequent increase in highly active hydroxyl radicals and other reactive oxygen species causing cellular damages. The GPx1 is one of the most important enzymes counteracting oxidative stress. In this work, we investigated a possible correlation between the GCG repeat polymorphism present in the first exon of GPx1 gene encoding a tract of five to seven alanine residues (ALA5, ALA6, and ALA7) and ASD. Our findings highlighted a high frequency of ALA5 allele in ASD subjects. Moreover, proteins corresponding to the three GPx1 variants were produced in vitro, and the evaluation of their activity showed a lower values for GPx1 having ALA5 polymorphism. The comparison of the secondary and tertiary structure predictions revealed an alpha-helix in correspondence of alanine stretch only in the case of GPx1-ALA7 variant. Finally, to better investigate protein structure, steady-state fluorescence measurements of GPx1 intrinsic tryptophan were carried out and the three tested proteins exhibited a different stability under denaturing conditions. This work demonstrates the importance in adopting a multidisciplinary strategy to comprehend the role of GPx1 in ASD

Polarized X-ray Absorption Near-Edge Structure Spectroscopy of Neuroglobin and Myoglobin Single Crystals

Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-neuroglobin (NgbCO) single crystals have been collected and compared with a number of derivatives of sperm whale myoglobin (Mb), that is, the nitrosyl (MbNO) and deoxy (Mb) derivatives, the previously reported cyanomet (MbCN) and carbonmonoxy (MbCO) derivatives, and the cryogenic photoproduct of MbCO (Mb center dot CO). The single crystals under study exhibit a strong XANES angular dichroism which allows the heme geometry of each sample to be analyzed with extremely high accuracy via the full multiple scattering (MS) approach. The results of two alternative methods to undergo the MS analysis have been compared with high resolution X-ray diffraction (XRD) data and with X-ray absorption spectroscopy (XAS) data in solution. As a result of the present analysis, the Fe-heme Structure in solution and in the cryo-trapped NgbCO single crystal (which cracks at room temperature) are the same. Accordingly, the residual energy involved in the protein relaxation responsible of crystal cracking at room temperature alter CO binding does not reside in the helm pocket. A combined approach (polarized XANES and XRD) is suggested to be applied on the same single crystals of metalloproteins at opportunely equipped synchrotron beamlines