CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

Summary The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex co-purifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5’ ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a ‘sea worm’ and is composed of a Cmr2-3 heterodimer ‘tail’, a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled ‘head’ containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. While Type I and Type II CRISPR-Cas surveillance complexes target double-stranded DNA, Type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy (cryo-EM) reconstructions of native Type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumb-like β-hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nt intervals. The Cmr complex is architecturally similar to the Type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.Bacteria and archaea defend themselves against infection using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and CRISPR-associated (Cas) genes (1). A defining feature of CRISPR-Cas systems is the use of Cas proteins in complex with small CRISPR RNAs (crRNAs) to identify and cleave The effector complex of the Type III system from T. thermophilus (Cmr) is a 12-subunit assembly composed of six Cmr subunits (Cmr1-6) and a crRNA with a stoichiometry of Cmr1 1 2 1 3 1 4 4 5 3 6 1 :crRNA 1 (7). The Cmr complex binds to target RNA that is complementary to the bound 40 or 46-nt crRNA and cleaves the target at 6-nt intervals measured from the 5' end of the crRNA sequence (7,8). Although low-resolution structural studies revealed an overall capsule-like architecture of the Cmr complex (7), the molecular basis of subunit assembly, crRNA binding and ssRNA target recognition and cleavage by the intact surveillance complex remains unknown.We performed cryo-electron microscopy (cryo-EM) of the intact ~350-kDa Cmr complex in the absence and presence of target ssRNA. We purified endogenous apo-Cmr (containing a crRNA) and used this sample for step-wise assembly with a 56-nt biotinylated ssRNA target followed by purification using streptavidin affinity chromatography. Frozen-hydrated samples of both apo-Cmr and target-bound Cmr were visualized using an FEI Titan Krios microscope equipped with a Gatan K2 Summit direct electron detector. Cryo-EM micrographs of both apo-Cmr and the ssRNA-bound complex showed mono-disperse, easily identifiable particles with sea worm-like features ( fig. S1). Using LEGINON (9), we acquired ~7,000 and ~4,000 micrographs and automatically picked ~700,000 and ~300,000 apo-and target-bound Cmr particles, respectively, using Appion (10). After 3D classification and single-particle reconstruction (Supplementary Material and Methods) in RELION (11), we obtained structures of intact apo-Cmr and target-bound Cmr at ~4.1 and 4.4-Å resolution ( fig. S1, S2) (using the 0.143 gold standard Fourier Shell Correlationcalc...

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The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH 2 -terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH 2 -terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.

The extremely thermophilic bacterium Thermus thermophilus HB8, which belongs to the phylum DeinococcusThermus, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named T. thermophilus CRP is highly homologous to the CRP family proteins from the phyla Firmicutes, Actinobacteria, and Cyanobacteria, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this strain, which did not drastically fluctuate during cultivation in a rich medium. The expression of several genes was altered upon disruption of the T. thermophilus CRP gene. We found six CRP-cAMP-dependent promoters in in vitro transcription assays involving DNA fragments containing the upstream regions of the genes exhibiting de- Cyclic AMP (cAMP) receptor proteins (CRPs) are global transcriptional regulators broadly distributed in bacteria (30,72). The cellular roles of such CRP family proteins are diverse and include carbohydrate metabolism (3, 30), development of competence for transformation (8), modulation of virulence gene expression and pathogenesis (10,11,55,57,65), resuscitation (50), and germination and morphological development (13,49).Escherichia coli CRP controls the activity of over 100 genes and has been the most extensively studied so far (30, 72). This CRP was first named the catabolite gene-activating protein, since it induces the transcription of a number of genes in response to carbon source limitation (16,73). In the absence of a carbon source such as glucose, the intracellular cAMP level increases, resulting in the formation of a CRP-cAMP complex, which binds to specific DNA sequences at target promoters. The CRP-cAMP regulatory complex is also involved in the regulation of genes that are not directly related to catabolism (3). In addition, the complex acts as a negative regulator of transcription at cya gene promoter cyaP2, gal operon promoter galP2, crp gene promoter crpP, and deo operon promoter deoP2 (3). E. coli CRP is a dimer of two identical subunits, each 209 residues in length, and contains a helix-turn-helix DNA-binding motif in its C-terminal domain (40). Each subunit can bind one molecule of allosteric effector cAMP. This CRP undergoes a conformational change upon cAMP binding (21,66,67), and the CRP-cAMP complex interacts with a 22-bp DNA site exhibiting twofold symmetry, with the consensus sequence 5Ј-AAATGTGATCTAGATCACATTT-3Ј (15). Biochemical and genetic analyses have revealed that this CRP interacts with the C-terminal domain of the RNA polymerase (RNAP) ␣ subunit (␣CTD) (5,6,24,35,43,44,58). This interaction is thought to facilitate RNAP binding to the promoter, which leads to the formation of an open complex and induction of transcription initiation. Crystallographic studies on E. coli CRP have been performed to determine the structure of CRP-cAMP and the mechanisms underlying the interactions among CRP-cAMP, DNA, and RNAP ␣CTD (30, 34).CRP homologs have been found not only in othe...