Neuroglobin is a member of the globin family involved in neuroprotection; it is primarily expressed in the brain and retina of vertebrates. Neuroglobin belongs to the heterogeneous group of hexacoordinate globins that have evolved in animals, plants and bacteria, endowed with the capability of reversible intramolecular coordination, allowing the binding of small gaseous ligands (O2, NO and CO). In a unique fashion among haemoproteins, ligand-binding events in neuroglobin are dependent on the sliding of the haem itself within a preformed internal cavity, as revealed by the crystal structure of its CO-bound derivative. Point mutants of the neuroglobin internal cavity have been engineered and their functional and structural characterization shows that hindering the haem displacement leads to a decrease in CO affinity, whereas reducing the cavity volume without interfering with haem sliding has negligible functional effects.

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 human DPM1 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.

The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E–eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

The 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.time-resolved X-ray scattering | protein conformational changes | cooperativity | flash photolysis E ver since the publication of the Monod-Wyman-Changeux paper on allostery (1), hemoglobin (Hb) has been considered the prototype of an allosteric protein; the molecular basis of positive cooperativity in O 2 binding involving a ligand-linked shift between two different quaternary states. The dynamics of ligand rebinding and of the tertiary and quaternary allosteric changes of tetrameric human Hb have been investigated, by-and-large, using transient spectroscopy in the picosecond to millisecond time range, following laser-induced photolysis of the ligand-heme iron bond. Starting with the carbon monoxide adduct HbCO in the allosteric quaternary state called R 4 , complete photolysis yields the unliganded R 0 state; the destiny of this photoproduct is a complex time-dependent process involving competing events such as ligand rebinding and (tertiary and quaternary) conformational decays. Changes in the optical and resonance Raman spectra of the different states have provided, over the last four decades, a quantitative estimate of the rates of the competing events (2-5). For a review on time-resolved optical absorption (TR-OA) data describing conformational decays as well as rebinding in the dark of a ligand...

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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·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 after CO binding does not reside in the heme 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.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV–visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15–40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

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