Keiko Sakamoto

RNA Targeting by the Type III-A CRISPR-Cas Csm Complex of Thermus thermophilus

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.

Transcription profile of Thermus thermophilus CRISPR systems after phage infection.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage PhiYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.e., from the eclipse period to the period of host cell lysis). Interestingly, the expression of most cAMP receptor protein (CRP)-regulated genes, including two CRISPR-associated (cas) operons, was most markedly up-regulated, especially around the beginning of host cell lysis, although up-regulation of the crp gene was not observed. The expression of the CRP-regulated genes was less up-regulated in a crp-deficient strain than in the wild type. Thus, it is suggested that cAMP is a signaling molecule that transmits information on phage infection to CRP to up-regulate these genes. On the other hand, the expression of several cas genes and that of CRISPRs were up-regulated independent of CRP, suggesting the involvement of unidentified regulatory factor(s) induced by phage infection. On analysis of the expression profile of the entire genome, we could speculate that upon phage infection, the signal was transmitted to the cells, with host response systems including CRISPR defense systems being activated, while the overall efficiencies of transcription, translation, and metabolism in the cells decreased. These findings will facilitate understanding of the host response mechanism following phage infection.

Structural and functional characterization of the transcriptional repressor CsoR from Thermus thermophilus HB8

The TTHA1719 gene from Thermus thermophilus HB8 encodes an orthologue of the copper-sensing transcriptional repressor CsoR. X-ray crystal structure analysis of T. thermophilus CsoR indicated that it forms a homotetramer. The structures of the CsoR monomer and dimer are similar to those of Mycobacterium tuberculosis CsoR. In the absence of copper ions, T. thermophilus CsoR bound to the promoter region of the copper-sensitive operon copZ-csoR-copA, which encodes the copper chaperone CopZ, CsoR and the copper efflux P-type ATPase CopA, to repress their expression, while in the presence of approximately an equal amount of copper ion, CsoR was released from the DNA, to allow expression of the downstream genes. Both Cu(II) and Cu(I) ions could bind CsoR, and were effective for transcriptional derepression. Additionally, CsoR could also sense various other metal ions, such as Zn(II), Ag(I), Cd(II) and Ni(II), which led to transcriptional derepression. The copper-binding motif of T. thermophilus CsoR contains C-H-H, while those of most orthologues contain C-H-C. The X-ray crystal structure of T. thermophilus CsoR suggests that a histidine residue in the N-terminal domain is also involved in metal-ion binding; that is, the binding motif could be H-C-H-H, like that of Escherichia coli RcnR, which binds Ni(II)/Co(II). The non-conserved H70 residue in the metal-binding motif of T. thermophilus CsoR is important for its DNA-binding affinity and metal-ion responsiveness.

TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation.

In the extremely thermophilic bacterium Thermus thermophilus HB8, one of the four TetR-family transcriptional regulators, which we named T. thermophilus FadR, negatively regulated the expression of several genes, including those involved in fatty acid degradation, both in vivo and in vitro. T. thermophilus FadR repressed the expression of the target genes by binding pseudopalindromic sequences covering the predicted -10 hexamers of their promoters, and medium-to-long straight-chain (C10-18) fatty acyl-CoA molecules were effective for transcriptional derepression. An X-ray crystal structure analysis revealed that T. thermophilus FadR bound one lauroyl (C12)-CoA molecule per FadR monomer, with its acyl chain moiety in the centre of the FadR molecule, enclosed within a tunnel-like substrate-binding pocket surrounded by hydrophobic residues, and the CoA moiety interacting with basic residues on the protein surface. The growth of T. thermophilus HB8, with palmitic acid as the sole carbon source, increased the expression of FadR-regulated genes. These results indicate that in T. thermophilus HB8, medium-to-long straight-chain fatty acids can be used for metabolic energy under the control of FadR, although the major fatty acids found in this strain are iso- and anteiso-branched-chain (C15 and 17) fatty acids.

X-ray crystal structure of a CRISPR-associated RAMP superfamily protein, Cmr5, from Thermus thermophilus HB8

X-ray crystal structure of a CRISPRassociated RAMP superfamily protein, Cmr5, from Thermus thermophilus HB8 Keiko Sakamoto,1y Yoshihiro Agari,1y Kazuko Agari, Shigeyuki Yokoyama, Seiki Kuramitsu, and Akeo Shinkai* 1 RIKEN SPring-8 Center, Harima Institute, Sayo, Hyogo 679-5148, Japan 2 RIKEN Systems and Structural Biology Center, Yokohama Institute, Tsurumi, Yokohama 230-0045, Japan 3 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 4 Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

Crystal structure of glutamine receptor protein from Sulfolobus tokodaii strain 7 in complex with its effector l-glutamine: implications of effector binding in molecular association and DNA binding

Genome analyses have revealed that members of the Lrp/AsnC family of transcriptional regulators are widely distributed among prokaryotes, including both bacteria and archaea. These regulatory proteins are involved in cellular metabolism in both global and specific manners, depending on the availability of the exogenous amino acid effectors. Here we report the first crystal structure of glutamine receptor protein (Grp) from Sulfolobus tokodaii strain 7, in the ligand-free and glutamine-bound (Grp-Gln) forms. Although the overall structures of both molecules are similar, a significant conformational change was observed at the ligand [l-glutamine (Gln)] binding site in the effector domain, which may be essential for further stabilization of the octameric structure, and in turn for facilitating DNA binding. In addition, we predicted promoter for the grp gene, and these analyses suggested the importance of cooperative binding to the protein. To gain insights into the ligand-induced conformational changes, we mutated all of the ligand-binding residues in Grp, and revealed the importance of Gln binding by biochemical and structural analyses. Further structural analyses showed that Y77 is crucial for ligand binding, and that the residues T132 and T134, which are highly conserved among the Lrp family of proteins, fluctuates between the active and inactive conformations, thus affecting protein oligomerization for DNA binding.

Functional identification of an anti-σE factor from Thermus thermophilus HB8

The TTHB212 gene from extremely thermophilic bacterium Thermus thermophilus HB8 forms an operon with the upstream sigE gene encoding an extracytoplasmic function sigma factor, sigma(E), the sole alternative sigma factor of this strain, on megaplasmid pTT27. The TTHB212 gene encodes a poorly conserved protein, which has been predicted to be a transmembrane one with N-terminal intracellular and C-terminal extracytoplasmic domains. The N-terminal domain of TTHB212 protein (TTHB212N) prevented sigma(E) from binding to RNA polymerase (RNAP) core enzyme in vitro, and TTHB212N bound sigma(E) in a molar ratio of 1:1 when both proteins were co-expressed in Escherichia coli. Furthermore, TTHB212N inhibited the transcription activity of RNAP-sigma(E) holoenzyme, but not that of the RNAP-sigma(A) one, in vitro. The expression of several genes that are under the control of sigma(E) was increased in a TTHB212 gene-disruptant strain. Thus, TTHB212 protein was identified as an anti-sigma(E) factor. These findings indicate that T. thermophilus HB8 has a regulatory system involving sigma(E) and anti-sigma(E) factors.

Structure and function of a TetR family transcriptional regulator, SbtR, from thermus thermophilus HB8

SbtR is one of the four TetR family transcriptional regulators present in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified 10 genes controlled by four promoters with negative regulation by SbtR in vitro. The SbtR‐regulated gene products include probable transporters, probable enzymes for sugar or amino acid metabolism, and nucleic acid‐related enzymes. SbtR binds pseudopalindromic sequences, with the consensus sequence of 5′‐TGACCCNNKGGTCA‐3′ surrounding the promoters, and has a proposed 1:1 dimer binding stoichiometry. The X‐ray crystal structure analysis revealed that SbtR comprises either nine or 10 α‐helices and forms a dimer, as in the typical TetR family proteins. Similar to many characterized TetR family regulators, SbtR has a predicted ligand‐binding pocket at the center of each monomer. Interestingly, the SbtR dimer contains an intermolecular disulfide bridge, formed between the Cys164 residues at the entrance of the pocket. The Cys164Ser and Cys164Ala mutant SbtR proteins formed homodimers similar to that of the wild type, but their thermal stabilities were lower by about 8°C, indicating that the disulfide bridge contributes to the thermal stability of the protein. However, altered repression activity of the mutants was not observed in vitro. From these results, we propose that ligand‐binding is essential for SbtR to disengage from DNA, in a similar manner to the other characterized TetR family regulators. The formation and reduction of the disulfide bond might function in controlling the ligand‐binding affinity of this transcriptional regulator. Proteins 2013; 81:1166–1178.

Expression, purification, crystallization and preliminary X‐ray diffraction analysis of galactokinase from Pyrococcus horikoshii. Erratum

Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

Crystallization and preliminary crystallographic studies of L30e, a ribosomal protein from Methanocaldococcus jannaschii (MJ1044)

In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 x 0.20 x 0.35 mm) was obtained that belonged to the primitive tetragonal space group P4(3), with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 A, and diffracted to a resolution of 1.9 A. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (V(M)) of 2.16 A(3) Da(-1).