No abstract

Protein turnover is an essential process in living cells. The degradation of cytosolic polypeptides is mainly carried out by the proteasome, resulting in 7-9-amino acid long peptides. Further degradation is usually carried out by energy-independent proteases like the tricorn protease from Thermoplasma acidophilum. Recently, a novel tetrahedral-shaped dodecameric 480-kDa aminopeptidase complex (TET) has been described in Haloarcula marismortui that differs from the known ring-or barrel-shaped self-compartmentalizing proteases. This complex is capable of degrading most peptides down to amino acids. We present here the crystal structure of the tetrahedral aminopeptidase homolog FrvX from Pyrococcus horikoshii. The monomer has a typical clan MH fold, as found for example in Aeromonas proteolytica aminopeptidase, containing a dinuclear zinc active center. The quaternary structure is built by dimers with a length of 100 Å that form the edges of the tetrahedron. All 12 active sites are located on the inside of the tetrahedron. Substrate access is granted by pores with a maximal diameter of 10 Å, allowing only small peptides and unfolded proteins access to the active site.The protein content of all living cells is constantly renewed through synthesis of new proteins and degradation of unneeded or misfolded proteins. This catabolism of proteins is a key cellular function and must be under spatial and temporal control to avert damage to the cell. Because prokaryotes lack membrane-bound compartments, degradation mostly takes place in large macromolecular self-compartmentalizing assemblies whose active sites are arranged in an inner cavity in order to only allow proteolysis of unfolded substrates.In all three kingdoms the degradation of cytosolic proteins is carried out predominantly by the ATP-dependent proteasome or similar energy-dependent proteases that generate oligopeptides 7-9 amino acids long (1-3). These products are thought to be further processed by assemblies like the energy-independent tricorn protease found in Thermoplasma acidophilum (4 -6). Other large energy-independent protease complexes that putatively take part in the degradation of oligopeptides created by the proteasome are the mammalian TPPII protease (7), the DppA D-aminopeptidase from Bacillus subtilis (8), and yeast bleomycin hydrolase (9). All these multimeric complexes are metalloenzymes and are composed of rings or barrels with a single central channel and only two openings.Recently, a novel protease complex called tetrahedral aminopeptidase (TET) 1 has been isolated from the Archaea Haloarcula marismortui (10). Electron microscopy analysis of TET at 17 Å resolution showed that it is a 0.4-MDa homododecameric complex with a novel tetrahedral shape that is made up by association of six antiparallel dimers. Contrary to all other self-compartmentalizing proteases described before, the central cavity of this complex is accessible through four narrow channels and through four wider channels. By spectrophotometric assays TET was shown to have aminopep...

BackgroundThe serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.Principal FindingsHere we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.SignificanceThe structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

The metzincins constitute a subclan of metalloproteases possessing a HEXXHXXGXXH/D zinc-binding consensus sequence where the three histidines are zinc ligands and the glutamic acid is the catalytic base. A completely conserved methionine is located downstream of this motif. Families of the metzincin clan comprise, besides others, astacins, adamalysins proteases, matrix metalloproteases, and serralysins. The latter are extracellular 50 kDa proteases secreted by Gram-negative bacteria via a type I secretion system. While there is a large body of structural and biochemical information available, the function of the conserved methionine has not been convincingly clarified yet. Here, we present the crystal structures of a number of mutants of the serralysin member protease C with the conserved methionine being replaced by Ile, Ala, and His. Together with our former report on the leucine and cysteine mutants, we demonstrate here that replacement of the methionine side chain results in an increasing distortion of the zinc-binding geometry, especially pronounced in the x 2 angles of the first and third histidine of the consensus sequence. This is correlated with an increasing loss of proteolytic activity and a sharp increase of flexibility of large segments of the polypeptide chain.

Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 Å , respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of ϳ30 Å , which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.