The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.

Building protein structure-specific rotamer libraries.

MOTIVATION: Identifying the probable positions of the protein side-chains is one of the protein modelling steps that can improve the prediction of protein-ligand and protein-protein interactions. Most of the strategies predicting the side-chain conformations use predetermined dihedral angle lists, also called rotamer libraries, that are usually generated from a subset of high-quality protein structures. Although these methods are fast to apply, they tend to average out geometries instead of taking into account the surrounding atoms and molecules and ignore structures not included in the selected subset. Such simplifications can result in inaccuracies when predicting possible side-chain atom positions. RESULTS: We propose an approach that takes into account both of these circumstances by scanning through sterically accessible side-chain conformations and generating dihedral angle libraries specific to the target proteins. The method avoids the drawbacks of lacking conformations due to unusual or rare protein structures and successfully suggests potential rotamers with average RMSD closer to the experimentally determined side-chain atom positions than other widely used rotamer libraries. AVAILABILITY AND IMPLEMENTATION: The technique is implemented in open-source software package rotag and available at GitHub: https://www.github.com/agrybauskas/rotag, under GNU Lesser General Public License.

PLBD: protein-ligand binding database of thermodynamic and kinetic intrinsic parameters.

We introduce a protein-ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein-ligand crystal structures, enabling structure-thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein-ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein-ligand recognition and could be integrated into small molecule drug design. Database URL https://plbd.org/.

Structural basis for sequence-specific recognition of guide and target strands by the Archaeoglobus fulgidus Argonaute protein.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like 'long' and truncated 'short' pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5'-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.

Graph isomorphism-based algorithm for cross-checking chemical and crystallographic descriptions.

Published reports of chemical compounds often contain multiple machine-readable descriptions which may supplement each other in order to yield coherent and complete chemical representations. This publication presents a method to cross-check such descriptions using a canonical representation and isomorphism of molecular graphs. If immediate agreement between compound descriptions is not found, the algorithm derives the minimal set of simplifications required for both descriptions to arrive to a matching form (if any). The proposed algorithm is used to cross-check chemical descriptions from the Crystallography Open Database to identify coherently described entries as well as those requiring further curation.

Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion.

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.

Domain organization of DNase from Thioalkalivibrio sp. provides insights into retention of activity in high salt environments.

Our study indicates that DNA binding domains are common in many halophilic or halotolerant bacterial DNases and they are potential activators of enzymatic activity at high ionic strength. Usually, proteins adapt to high ionic strength by increasing the number of negatively charged residues on the surface. However, in DNases such adaptation would hinder the binding to negatively charged DNA, a step critical for catalysis. In our study we demonstrate how evolution has solved this dilemma by engaging the DNA binding domain. We propose a mechanism, which enables the enzyme activity at salt concentrations as high as 4 M of sodium chloride, based on collected experimental data and domain structure analysis of a secreted bacterial DNase from the extremely halotolerant bacterium Thioalkalivibrio sp. K90mix. The enzyme harbors two domains: an N-terminal domain, that exhibits DNase activity, and a C-terminal domain, comprising a duplicate DNA binding helix-hairpin-helix motif. Here we present experimental data demonstrating that the C-terminal domain is responsible for the enzyme's resistance to high ionic strength.