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Michael Groll

Publications and source records attributed to Michael Groll.

26 records · Page 2Linked to original sources
PubMed2003

Investigations on the maturation and regulation of archaebacterial proteasomes.

The 20S proteasome (core particle, CP) is a multifunctional protease complex and composed of four heptameric subunit rings arranged in a hollow, barrel-shaped structure. Here, we report the crystal structure of the CP from Archaeoglobus fulgidus at 2.25A resolution. The analysis of the structure of early and late assembly intermediates of this CP gives new insights in the maturation of archaebacterial CPs and indicates similarities to assembly intermediates observed in eukaryotes. We also show a striking difference in mechanism and regulation of substrate access between eukaryotic and archaebacterial 20S proteasomes. While eukaryotic CPs are auto-inhibited by the N-terminal tails of the outer alpha-ring by imposing topological closure with a characteristic sequence motif (YDR-motif) and show regulatory gating this segment is disordered in the CP and differently structured in the alpha(7)-sub-complex of A.fulgidus leaving a pore leading into the particle with a diameter of 13A. Mutagenesis and functional studies indicate the absence of regulatory gating in the archaeal 20S proteasome.

Amino Acid Sequence↗
PubMed2003

Molecular shredders: how proteasomes fulfill their role.

The 20S proteasome is a large, cylinder-shaped protease that is found in all domains of life and plays a crucial role in cellular protein turnover. It has multiple catalytic centers located within the hollow cavity of a molecular cage. This architecture prevents unwanted degradation of endogenous proteins and promotes processive degradation of substrates by restricting the dissociation of partially digested polypeptides. Although this kind of self-compartmentalization is generally conserved, the proteasomes of bacteria, archaea and eukaryotes show many differences in architecture, subunit composition and regulation. The structure of the 20S proteasome and its inherent role in the regulation of proteasome function are gradually being elucidated.

Animals↗
PubMed2003

Substrate access and processing by the 20S proteasome core particle.

Intracellular proteolysis is an essential process. In eukaryotes, most proteins in the cytosol and nucleus are degraded by the ubiquitin (Ub)-proteasome pathway. A major component within this system is the 26S proteasome, a 2.5MDa molecular machine, built from more than 31 different subunits. This complex is formed by a cylinder-shaped multimeric complex referred to as the proteolytic 20S proteasome (core particle, CP) capped at each end by another multimeric component called the 19S complex (regulatory particle, RP) or PA700. Structure, assembly and enzymatic mechanism have been elucidated only for the CP, whereas the organization of the RP is less well understood. The CP is composed of 28 subunits, which are arranged as an alpha7beta7beta7alpha7-complex in four stacked rings. The interior of the free core particle, which harbors the active sites, is inaccessible for folded and unfolded substrates and represents a latent state. This inhibition is relieved upon binding of the RP to the CP by formation of the 26S proteasome holoenzyme. This review summarizes the current knowledge of the structural features of 20S proteasomes.

Animals↗
PubMed2002

Navigation inside a protease: substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum.

The proposed pathway and mechanism of substrate entry and product egress in the hexameric D3 symmetric tricorn protease from Thermoplasma acidophilum were explored by crystallographic studies of ligand complexes and by structure-based mutagenesis. Obstruction of the pore within the 7-bladed beta-propeller (beta7) domain by alkylation or oxidation of an engineered double cysteine mutant strongly decreased enzymatic activities. In line herewith, the crystal structure of the tricorn protease in complex with a trideca-peptide inhibitor modifying the catalytic Ser965 revealed part of the peptide trapped inside the channel of the beta7 domain. The cysteine mutation widening the lumen of the 6-bladed beta-propeller (beta6) domain enhanced catalytic activity, which was restored to normal values after its alkylation. A charge reversal mutant at the putative anchor site of the substrate C terminus, R131E-R132E, drastically reduced the proteolytic activity. The complex crystal structure of a peptide inhibitor with a diketo group at the cleavage site mapped the substrate recognition site and confirmed the role of Arg131-Arg132 as an anchor site. Our results strongly suggest the wider beta7 domain to serve as a selective filter and guide of the substrate to the sequestered active site, while the narrower beta6 domain routes the product to the surface. Moreover, we identified the role of Arg131-Arg132 in anchoring the substrate C terminus.

Alkylation↗
PubMed2002

Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism.

F1 is a 33.5 kDa serine peptidase of the alpha/beta-hydrolase family from the archaeon Thermoplasma acidophilum. Subsequent to proteasomal protein degradation, tricorn generates small peptides, which are cleaved by F1 to yield single amino acids. We have solved the crystal structure of F1 with multiwavelength anomalous dispersion (MAD) phasing at 1.8 A resolution. In addition to the conserved catalytic domain, the structure reveals a chiefly alpha-helical domain capping the catalytic triad. Thus, the active site is accessible only through a narrow opening from the protein surface. Two structures with molecules bound to the active serine, including the inhibitor phenylalanyl chloromethylketone, elucidate the N-terminal recognition of substrates and the catalytic activation switch mechanism of F1. The cap domain mainly confers the specificity for hydrophobic side chains by a novel cavity system, which, analogously to the tricorn protease, guides substrates to the buried active site and products away from it. Finally, the structure of F1 suggests a possible functional complex with tricorn that allows efficient processive degradation to free amino acids for cellular recycling.

Amino Acid Sequence↗
PubMed2002

Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome.

The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.

Binding Sites↗
PubMedDate not supplied

Structural basis for the processive protein degradation by tricorn protease.

Cell survival critically depends on the efficient use of available resources. This includes both the clearance and the recycling of those protein components that have become futile or defective. Several proteins sequentially accomplish this complex task. The proteasome serves as an initial protein shredder and generates peptides of 7-12 amino acids in length. In general, these products are useless burden to the cell and need further processing. A few years ago, a proteolytic system was identified in the model organism Thermoplasma acidophilum which indeed performs this processing [Tamura et al., Science 274 (1996), 1385-1389]. The hexameric core protein of this modular system, referred to as tricorn protease, is a 720 kDa protease which is able to assemble further into a giant icosahedral capsid, as determined by electron microscopy. Recently, we determined the crystal structure of the tricorn core particle at 2.0 A resolution [Brandstetter et al., Nature 414 (2001), 466-469]. Here we describe the structural and mechanistic basis for tricorn's processive degradation mode, including a novel electrostatic substrate-to-product sink, and suggest how further components might interact with the tricorn protease to complete the cellular waste recycling process.

Archaeal Proteins↗