Francesca Germani

Structure-Based Inhibition of Norovirus RNA-Dependent RNA Polymerases.

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.

Co Rebinding Kinetics and Molecular Dynamics Simulations Highlight Dynamic Regulation of Internal Cavities in Human Cytoglobin.

Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein.

A redox signalling globin is essential for reproduction in Caenorhabditis elegans

Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers.

Globins in Caenorhabditis elegans

Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell‐specific expression patterns and fixation into the genome. To date, four globins (GLB‐1, GLB‐5, GLB‐6, and GLB‐26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB‐1 and GLB‐6) was solved. In this article, a three‐dimensional model was designed for the other two globins (GLB‐5 and GLB‐26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three‐dimensional structure have major implications on their ligand‐binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions.

Haem-Based Sensors: A Still Growing Old Superfamily

The haem-based sensors are chimeric multi-domain proteins responsible for the cellular adaptive responses to environmental changes. The signal transduction is mediated by the sensing capability of the haem-binding domain, which transmits a usable signal to the cognate transmitter domain, responsible for providing the adequate answer. Four major families of haem-based sensors can be recognized, depending on the nature of the haem-binding domain: (i) the haem-binding PAS domain, (ii) the CO-sensitive carbon monoxide oxidation activator, (iii) the haem NO-binding domain, and (iv) the globin-coupled sensors. The functional classification of the haem-binding sensors is based on the activity of the transmitter domain and, traditionally, comprises: (i) sensors with aerotactic function; (ii) sensors with gene-regulating function; and (iii) sensors with unknown function. We have implemented this classification with newly identified proteins, that is, the Streptomyces avermitilis and Frankia sp. that present a C-terminal-truncated globin fused to an N-terminal cofactor-free monooxygenase, the structural-related class of non-haem globins in Bacillus subtilis, Moorella thermoacetica, and Bacillus anthracis, and a haemerythrin-coupled diguanylate cyclase in Vibrio cholerae. This review summarizes the structures, the functions, and the structure-function relationships known to date on this broad protein family. We also propose unresolved questions and new possible research approaches.

High Resolution Crystal Structures of the Cerebratulus Lacteus Mini-Hb in the Unligated and Carbomonoxy States.

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O2 dissociation and ultra-high O2 affinity. However, CerHb affinity for O2 is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O2 ligand, the overall binding schemes for CO and O2 are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: Molecular Modeling and Functional Properties

Background: GLB-33 is a putative neuropeptide receptor in C. elegans with a globin domain. Results: Recombinant globin domain displays a ferric hydroxide-ligated form. When reduced, it can bind CO or O2 and reduce nitrite to NO. Conclusion: The globin domain may serve as an oxygen sensor or nitrite reductase. Significance: Oxygen-sensing mechanisms are relevant for neuropeptide receptor binding. We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.