Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

Abstractl-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH3) and hydrogen peroxide (H2O2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1Å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62–71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode

Stability of spontaneous, correlated activity in mouse auditory cortex

Neural systems can be modeled as networks of functionally connected neural elements. The resulting network can be analyzed using mathematical tools from network science and graph theory to quantify the system's topological organization and to better understand its function. While the network-based approach is common in the analysis of large-scale neural systems probed by non-invasive neuroimaging, few studies have used network science to study the organization of networks reconstructed at the cellular level, and thus many very basic and fundamental questions remain unanswered. Here, we used two-photon calcium imaging to record spontaneous activity from the same set of cells in mouse auditory cortex over the course of several weeks. We reconstruct functional networks in which cells are linked to one another by edges weighted according to the correlation of their fluorescence traces. We show that the networks exhibit modular structure across multiple topological scales and that these multi-scale modules unfold as part of a hierarchy. We also show that, on average, network architecture becomes increasingly dissimilar over time, with similarity decaying monotonically with the distance (in time) between sessions. Finally, we show that a small fraction of cells maintain strongly-correlated activity over multiple days, forming a stable temporal core surrounded by a fluctuating and variable periphery. Our work provides a careful methodological blueprint for future studies of spontaneous activity measured by two-photon calcium imaging using cutting-edge computational methods and machine learning algorithms informed by explicit graphical models from network science. The methods are easily extended to additional datasets, opening the possibility of studying cellular level network organization of neural systems and how that organization is modulated by stimuli or altered in models of disease.Comment: 15 pages, 3 figure

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galβ1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66 kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62 Å resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the π electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-α-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same

Subtilisin BPN' at 1.6 Å Resolution: Analysis for Discrete Disorder and Comparison of Crystal Forms

The three-dimensional structure of the serine protease subtilisin BPN' (SBT) has been refined at 1.6 Å resolution in space group C2 to a final R value of 0.17. 17 regions of discrete disorder have been identified and analyzed. Two of these are dual-conformation peptide units; the remainder involve alternate rotamers of side chains either alone or in small clusters. The structure is compared with previously reported high-resolution models of SBT in two other space groups, P212121 and P21. Apart from the surface, there are no significant variations in structure among the three crystal forms. Structural variations observed at the protein surface occur predominantly in regions of protein-protein contact. The crystal packing arrangements in the three space groups are compared

Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations

Media pluralism: What matters for governance and regulation?

Media pluralism is valued in most jurisdictions because it contributes to a well-informed citizenry. The authors examine what media policy and regulatory levers appear to affect five types of citizen knowledge across the European Union. They conclude that concentration of titles matters more than ownership in newsprint; and that neither type of concentration matters in broadcasting in the same way, but the regulatory regime for public service broadcasting does, particularly for political knowledge

Real-time investigation of dynamic protein crystallization in living cells

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitroL.R., M.K., D.R., and C.B. thank the German Federal Ministry for Education and Research (BMBF) for funding (Grant Nos. 01KX0806 and 01KX0807). L.R., M.D., and C.B. acknowledge support from the BMBF in the context of the Röntgen-Angström-Cluster (Grant No. 05K12GU3). J.M.-C. and A.B.-N. acknowledge support from the Spanish Ministerio Economía y Competitividad (MINECO, Grant No. BFU2013-43513-R). I.V.M., R.D., and L.R. are grateful for support from the DFG Cluster of Excellence “Inflammation at Interfaces” (EXC 306)S

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site