CovidGraph: a graph to fight COVID-19.

SUMMARY: Reliable and integrated data are prerequisites for effective research on the recent coronavirus disease 2019 (COVID-19) pandemic. The CovidGraph project integrates and connects heterogeneous COVID-19 data in a knowledge graph, referred to as 'CovidGraph'. It provides easy access to multiple data sources through a single point of entry and enables flexible data exploration. AVAILABILITY AND IMPLEMENTATION: More information on CovidGraph is available from the project website: https://healthecco.org/covidgraph/. Source code and documentation are provided on GitHub: https://github.com/covidgraph. SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online.

Aligning, analyzing, and visualizing sequences for antibody engineering: Automated recognition of immunoglobulin variable region features.

The analysis and comparison of large numbers of immunoglobulin (Ig) sequences that arise during an antibody selection campaign can be time-consuming and tedious. Typically, the identification and annotation of framework as well as complementarity-determining regions (CDRs) is based on multiple sequence alignments using standardized numbering schemes, which allow identification of equivalent residues among different family members but often necessitate expert knowledge and manual intervention. Moreover, due to the enormous length variability of some CDRs the benefit of conventional Ig numbering schemes is limited and the calculation of correct sequence alignments can become challenging. Whereas, in principle, a well established set of rules permits the assignment of CDRs from the amino acid sequence alone, no currently available sequence alignment editor provides an algorithm to annotate new Ig sequences accordingly. Here we present a unique pattern matching method implemented into our recently developed ANTICALIgN editor that automatically identifies all hypervariable and framework regions in experimentally elucidated antibody sequences using so-called "regular expressions." By combination of this widely supported software syntax with the unique capabilities of real-time aligning, editing and analyzing extended sets of amino acid and/or nucleotide sequences simultaneously on a local workstation, ANTICALIgN provides a powerful utility for antibody engineering. Proteins 2016; 85:65-71. © 2016 Wiley Periodicals, Inc.

Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion.

To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT(L417M), as proven by SDS-PAGE and size exclusion chromatography. The bifunctional enzyme revealed beneficial kinetics over the two-component system, in particular at low substrate concentration.

ANTICALIgN: visualizing, editing and analyzing combined nucleotide and amino acid sequence alignments for combinatorial protein engineering.

ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.

Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity.

The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM  = 54.1 µM-1  Min-1 (KM  = 32 ± 3 µM; kcat  = 1,730 ± 39 Min-1 ), almost the same as the wild-type enzyme for NADH (kcat /KM  = 59.9 µM-1  Min-1 ; KM  = 14 ± 2 µM; kcat  = 838 ± 21 Min-1 ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM  = 3 µM-1  Min-1 ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP+ and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP+ and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

Developability assessment during the selection of novel therapeutic antibodies.

Therapeutic antibodies and antibody derivatives comprise the majority of today's biotherapeutics. Routine methods to generate novel antibodies, such as immunization and phage-display, often give rise to several candidates with desired functional properties. On the contrary, resource-intense steps such as the development of a cell line, a manufacturing process, or a formulation, are typically carried out for only one candidate. Therefore, "developability," that is, the likelihood for the successful development of a lead candidate into a stable, manufacturable, safe, and efficacious drug, may be used as an additional selection criterion. Employing a set of small-scale, fast, and predictive tests addressing biochemical and biophysical features, as well as in vivo fate can help to identify a clinical candidate molecule with promising properties at an early stage of drug development. This article gives an overview of existing methods for developability testing and shows how these assays can be interlaced in the lead selection process.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel ß-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million.

The DARC site: a database of aligned ribosomal complexes.

The ribosome is a highly dynamic machine responsible for protein synthesis within the cell. Cryo-electron microscopy (cryo-EM) and X-ray crystallography structures of ribosomal particles, alone and in complex with diverse ligands (protein factors, RNAs and small molecules), have revealed the dynamic nature of the ribosome and provided much needed insight into translation and its regulation. In the past years, there has been exponential growth in the deposition of cryo-EM maps into the Electron Microscopy Data Bank (EMDB) as well as atomic structures into the Protein Data Bank (PDB). Unfortunately, the deposited ribosomal particles usually have distinct orientations with respect to one another, which complicate the comparison of the available structures. To simplify this, we have developed a Database of Aligned Ribosomal Complexes, the DARC site (http://darcsite.genzentrum.lmu.de/darc/), which houses the available cryo-EM maps and atomic coordinates of ribosomal particles from the EMDB and PDB aligned within a common coordinate system. An easy-to-use, searchable interface allows users to access and download >130 cryo-EM maps and >300 atomic models in the format of brix and pdb files, respectively. The aligned coordinate system substantially simplifies direct visualization of conformational changes in the ribosome, such as subunit rotation and head-swiveling, as well as direct comparison of bound ligands, such as antibiotics or translation factors.

Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.

No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. We used cryo-EM to visualize NGD intermediates resulting from binding of the Dom34-Hbs1 complex to stalled ribosomes. At subnanometer resolution, all domains of Dom34 and Hbs1 were identified, allowing the docking of crystal structures and homology models. Moreover, the close structural similarity of Dom34 and Hbs1 to eukaryotic release factors (eRFs) enabled us to propose a model for the ribosome-bound eRF1-eRF3 complex. Collectively, our data provide structural insights into how stalled mRNA is recognized on the ribosome and how the eRF complex can simultaneously recognize stop codons and catalyze peptide release.

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome.

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Šresolution. This map, together with a 6.1-Šmap of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

alpha-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel.

As translation proceeds, the nascent polypeptide chain passes through a tunnel in the large ribosomal subunit. Although this ribosomal exit tunnel was once thought only to be a passive conduit for the growing nascent chain, accumulating evidence suggests that it may in fact play a more active role in regulating translation and initial protein folding events. Here we have determined single-particle cryo-electron microscopy reconstructions of eukaryotic 80S ribosomes containing nascent chains with high alpha-helical propensity located within the exit tunnel. The maps enable direct visualization of density for helices as well as allowing the sites of interaction with the tunnel wall components to be elucidated. In particular regions of the tunnel, the nascent chain adopts distinct conformations and establishes specific contacts with tunnel components, both ribosomal RNA and proteins, that have been previously implicated in nascent chain-ribosome interaction.

Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.

The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined subnanometer-resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data, we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state, the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain, contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.