Biomedical subjects

M Bochtler

Publications and source records attributed to M Bochtler.

7 recordsLinked to original sources

PubMed2001

The quaternary arrangement of HslU and HslV in a cocrystal: a response to Wang, Yale.

Protease HslV and ATPase HslU form an ATP-dependent protease in bacteria. We have previously determined the structure of the components of this protease. In the case of HslU, the structure was derived from HslU-HslV cocrystals, combining phase information from MAD and the previously determined HslV model. Whereas the structures of the components were confirmed in detail by later structures, the quaternary arrangement of HslV and HslU was not reproduced in later crystal forms. In a recent communication to this journal, Wang attempted a reinterpretation of our original data to account for this difference. In response, we demonstrate that difference Pattersons, difference Fouriers, molecular replacement calculations, R factors, and omit maps all support our original analysis and prove that the suggested reinterpretation is false by these criteria. In particular, we show that our crystals are essentially untwinned and that only the originally reported quaternary arrangement of HslV and HslU particles is consistent with the experimental data. We finally demonstrate that Wang's newly introduced R(tpart) method to predict translational corrections for a subset of the unit cell contents is systematically flawed.

ATP-Dependent Proteases↗

PubMed2000

Mutational studies on HslU and its docking mode with HslV.

HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition/translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the "sensor arginine" (R393) and the "arginine finger" (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

ATP-Dependent Proteases↗

PubMed2000

The structures of HsIU and the ATP-dependent protease HsIU-HsIV.

The degradation of cytoplasmic proteins is an ATP-dependent process. Substrates are targeted to a single soluble protease, the 26S proteasome, in eukaryotes and to a number of unrelated proteases in prokaryotes. A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU) in Escherichia coli. Its protease component HslV shares approximately 20% sequence similarity and a conserved fold with 20S proteasome beta-subunits. HslU is a member of the Hsp100 (Clp) family of ATPases. Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV-the first structure of a complete set of components of an ATP-dependent protease. HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components. Instead, there is conformational flexibility and domain motion in HslU and a localized order-disorder transition in HslV. Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states. They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein, the prototype of an AAA-ATPase. A third, mostly alpha-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases.

ATP-Dependent Proteases↗

PubMed1999

The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study.

We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. beta1(Pre3), beta2(Pup1), and beta5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of beta6(Pre7) and beta7(Pre4) are intermediately processed to their final forms by beta2(Pup1) in the wild-type enzyme and by beta5(Pre2) and beta1(Pre3) in the beta2(Pup1) inactive mutants. A role of the propeptide of beta1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits beta3(Pup3), beta6(Pre7), and beta7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.

Catalytic Domain↗

PubMed1999

The proteasome.

Proteasomes are large multisubunit proteases that are found in the cytosol, both free and attached to the endoplasmic reticulum, and in the nucleus of eukaryotic cells. Their ubiquitous presence and high abundance in these compartments reflects their central role in cellular protein turnover. Proteasomes recognize, unfold, and digest protein substrates that have been marked for degradation by the attachment of a ubiquitin moiety. Individual subcomplexes of the complete 26S proteasome are involved in these different tasks: The ATP-dependent 19S caps are believed to unfold substrates and feed them to the actual protease, the 20S proteasome. This core particle appears to be more ancient than the ubiquitin system. Both prokaryotic and archaebacterial ancestors have been identified. Crystal structures are now available for the E. coli proteasome homologue and the T. acidophilum and S. cerevisiae 20S proteasomes. All three enzymes are cylindrical particles that have their active sites on the inner walls of a large central cavity. They share the fold and a novel catalytic mechanism with an N-terminal nucleophilic threonine, which places them in the family of Ntn (N terminal nucleophile) hydrolases. Evolution has added complexity to the comparatively simple prokaryotic prototype. This minimal proteasome is a homododecamer made from two hexameric rings stacked head to head. Its heptameric version is the catalytic core of archaebacterial proteasomes, where it is sandwiched between two inactive antichambers that are made up from a different subunit. In eukaryotes, both subunits have diverged into seven different subunits each, which are present in the particle in unique locations such that a complex dimer is formed that has six active sites with three major specificities that can be attributed to individual subunits. Genetic, biochemical, and high-resolution electron microscopy data, but no crystal structures, are available for the 19S caps. A first step toward a mechanistic understanding of proteasome activation and regulation has been made with the elucidation of the X-ray structure of the alternative, mammalian proteasome activator PA28.

Adenosine Triphosphatases↗

PubMed1997

Crystal structure of heat shock locus V (HslV) from Escherichia coli.

Heat shock locus V (HslV; also called ClpQ) is the proteolytic core of the ATP-dependent protease HslVU in Escherichia coli. It has sequence similarity with the beta-type subunits of the eukaryotic and archaebacterial proteasomes. Unlike these particles, which display 72-point symmetry, it is a dimer of hexamers with 62-point symmetry. The crystal structure of HslV at 3.8-A resolution, determined by isomorphous replacement and symmetry averaging, shows that in spite of the different symmetry of the particle, the fold and the contacts between subunits are conserved. A tripeptide aldehyde inhibitor, acetyl-Leu-Leu-norleucinal, binds to the N-terminal threonine residue of HslV, probably as a hemiacetal, relating HslV also functionally to the proteasomes of archaea and eukaryotes.

ATP-Dependent Proteases↗

PubMed1997

Structure of 20S proteasome from yeast at 2.4 A resolution.

The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

Acetylcysteine↗