Structure of the DNA-bound spacer capture complex of a Type II CRISPR-Cas system

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2–6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel

The structure of SgrAI bound to DNA; recognition of an 8 base pair target

The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca2+ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn2+, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI

Catalytic residues in hydrolases: analysis of methods designed for ligand-binding site prediction

The comparison of eight tools applicable to ligand-binding site prediction is presented. The methods examined cover three types of approaches: the geometrical (CASTp, PASS, Pocket-Finder), the physicochemical (Q-SiteFinder, FOD) and the knowledge-based (ConSurf, SuMo, WebFEATURE). The accuracy of predictions was measured in reference to the catalytic residues documented in the Catalytic Site Atlas. The test was performed on a set comprising selected chains of hydrolases. The results were analysed with regard to size, polarity, secondary structure, accessible solvent area of predicted sites as well as parameters commonly used in machine learning (F-measure, MCC). The relative accuracies of predictions are presented in the ROC space, allowing determination of the optimal methods by means of the ROC convex hull. Additionally the minimum expected cost analysis was performed. Both advantages and disadvantages of the eight methods are presented. Characterization of protein chains in respect to the level of difficulty in the active site prediction is introduced. The main reasons for failures are discussed. Overall, the best performance offers SuMo followed by FOD, while Pocket-Finder is the best method among the geometrical approaches

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-{\AA} resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5'-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-A resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5'-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis