Expression, purification, crystallization and preliminary crystallographic analysis of a deblocking aminopeptidase from<i>Pyrococcus horikoshii</i>

Porciero, Sophie; Receveur-Brechot, Véronique; Mori, Kazushige; Franzetti, Bruno; Roussel, Alain

doi:10.1107/s1744309105001910

Cited by 8 publications

(4 citation statements)

References 15 publications

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“…Cryoelectron Microscopy Revealed Tetrahedral and Octahedral Hollow Particles-In gel filtration, the PhTET1 protein yields two peaks with apparent molecular masses of 400 and 800 kDa, respectively (22). Analytical ultracentrifugation studies, performed on the partially purified complexes, showed that in addition to a particle with a sedimentation coefficient s 20,w of 15 S, i.e.…”

Section: Resultsmentioning

confidence: 99%

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An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Schoehn

Vellieux

Durá

et al. 2006

Journal of Biological Chemistry

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Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.Cytosolic proteolysis is a key cellular process. In addition to its role in amino acid metabolism, it is responsible for protein quality control and the adjustment of the turnover of regulatory molecules (1, 2). Initial proteolysis is carried out by nonspecific endoproteases (1). Through oligomerization, these enzymes form barrel-like cellular subcompartments to protect cytoplasmic proteins from unwanted cleavage; the active sites are situated in proteolytic chambers, only accessible to unfolded polypeptides. In Eukarya and Archaea, the 20 S proteasome is the core of this proteolytic system (3, 4). The proteins to be degraded are unfolded and translocated into the proteolytic complex by energy-dependent regulatory particles (5). Polypeptide degradation results in multiple endoproteolytic cleavage, yielding short peptides (6 -12 residues in length) (6). The final breakdown of these fragments is probably achieved by a collection of ATP-independent exopeptidases, which have been proposed to be essential components of the protein degradation system (7,8).Structural studies on the Tricorn protease complex, an archaeal carboxypeptidase (peptidyl-di/tripeptidase), have shown that some exopeptidases can also form multimeric ring structures, with the active sites sequestered within an internal chamber (9). This feature of compartmentalization was also recognized in the three-dimensional structures of three ATP-independent intracellular aminopeptidases: Gal6, leucine aminopeptidase, and the D-aminopeptidase DppA (10 -12). In eukaryotic cells, the tripeptidyl-peptidase II also forms a giant, well organized toroidal oligomeric structure (13). All these exopeptidases could function as scavengers of the oligopeptides generated by the ATP-dependent endoproteases (14).TET is another type of a large hollow peptidase complex. It was characterized initially in the halophilic Archaea Haloarcula marismortui (15). The complex has a tetrahedral shape and is a broad substrate specificity ...

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Section: Resultsmentioning

confidence: 99%

“…The expression of the PH0519 protein from P. horikoshii in Escherichia coli, its purification, and the crystallization conditions are described elsewhere (22).…”

Section: Protein Expression Purification and Crystallizationmentioning

confidence: 99%

An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Schoehn

Vellieux

Durá

et al. 2006

Journal of Biological Chemistry

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“…Hexahistidine-tagged proteins were expressed in E. Coli (BL21-Gold[DE3] pLysS; Stratagene) grown in 1 l of either Terrific Broth or selenomethionine medium [ 92 ] in the presence of 50 μg/ml kanamycin, 25 μg/ml chloramphenicol, and in the absence or presence of hemin (12.5 μM; Sigma. Cells were grown at 37 °C to an OD 600 of 1.2, and, following the addition of isopropyl-1-thio-D-galactopyranoside (final concentration 1 mM) to induce expression, the cells were incubated overnight with shaking at 25 °C.…”

Section: Methodsmentioning

confidence: 99%

The Structural Basis of Gas-Responsive Transcription by the Human Nuclear Hormone Receptor REV-ERBβ

et al. 2009

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Heme is a ligand for the human nuclear receptors (NR) REV-ERBα and REV-ERBβ, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 Å crystal structure of the REV-ERBβ LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBβ complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.

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“…As described in x2, we used crystallization conditions that led to a new high-resolution form of PhTET1-12s in space group P2 1 with an entire dodecamer in the asymmetric unit (Dura et al, 2010). Surprisingly, the addition of the tris-dipicolinate complex led to the initial F4 1 32 crystal form diffracting at low resolution, that was used for the initial structure determination of PhTET1-12s at 3.09 Å resolution (Porciero et al, 2005;Schoehn et al, 2006).…”

Section: Derivative Crystal Formmentioning

confidence: 99%