Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein.

A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

Structure and Function of the Bacterial Protein Toxin Phenomycin.

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin, however, have remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure that is critical for the toxicity of phenomycin.

Publisher Correction: Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity.

In the original version of this Article, molecular weight markers in Figs 1c, 2c,d and 4d were displaced during the production process, so that they were not correctly aligned with the corresponding bands. In addition, in Fig. 4c, molecular masses given for three different elution volumes were displaced so that they appeared to the left of the correct positions. These errors have now been corrected.

Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity.

Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but so far not in archaeal viruses, despite the near ubiquity of CRISPR-Cas systems in archaea. Here, we report the functional and structural characterization of two archaeal Acrs from the lytic rudiviruses, SIRV2 and SIRV3. We show that a 4 kb deletion in the SIRV2 genome dramatically reduces infectivity in Sulfolobus islandicus LAL14/1 that carries functional CRISPR-Cas subtypes I-A, I-D and III-B. Subsequent insertion of a single gene from SIRV3, gp02 (AcrID1), which is conserved in the deleted fragment, successfully restored infectivity. We demonstrate that AcrID1 protein inhibits the CRISPR-Cas subtype I-D system by interacting directly with Cas10d protein, which is required for the interference stage. Sequence and structural analysis of AcrID1 show that it belongs to a conserved family of compact, dimeric αß-sandwich proteins characterized by extreme pH and temperature stability and a tendency to form protein fibres. We identify about 50 homologues of AcrID1 in four archaeal viral families demonstrating the broad distribution of this group of anti-CRISPR proteins.

The 1.4-A crystal structure of the S. pombe Pop2p deadenylase subunit unveils the configuration of an active enzyme.

Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3'-5' exonucleases potentially responsible for the deadenylation reaction. Here, we present the crystal structure of the Pop2p subunit from Schizosaccharomyces pombe determined to 1.4 A resolution and show that the enzyme is a competent ribonuclease with a tunable specificity towards poly-A. In contrast to S. cerevisiae Pop2p, the S. pombe enzyme contains a fully conserved DEDDh active site, and the high resolution allows for a detailed analysis of its configuration, including divalent metal ion binding. Functional data further indicates that the identity of the ions in the active site can modulate both activity and specificity of the enzyme, and finally structural superposition of single nucleotides and poly-A oligonucleotides provide insight into the catalytic cycle of the protein.

Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain.

The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.

Digital photography: enhancing communication between burn therapists and nurses.

Burn rehabilitation therapists rely on nursing staff to follow through with the positioning and splinting programs. To communicate more effectively, a communication tool that consisted of digital photos and written instructions was created. Microsoft Word and Nikon View software were used to design the communication tool. The purpose of the study was to assess the perceived effectiveness of a communication tool between burn therapists and burn nurses for splinting and positioning. Thirty-two surveys were distributed to burn nursing staff to assess their perception of the communication tool (digital photographs with written instructions) compared with previous methods of instructions (without digital photographs). Seventy-three percent of nurses felt the communication tool with verbal instructions were the best methods of communicating splinting and positioning needs. All respondents felt that the rehabilitation staff should continue to use the communication tool.