An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Abstract

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 ...

Three-Dimensional Structure of Canine Adenovirus Serotype 2 Capsid

“…Four microliters of the recombinant CAV-2 fiber sample (ϳ0.1 mg/ml) was loaded in the mica-carbon interface as described previously (46). The sample was stained using 2% sodium silico tungstate, pH 7.5, and air dried.…”

Pyrococcus horikoshii TET2 Peptidase Assembling Process and Associated Functional Regulation

“…The TET particle interior is accessible via the openings situated on each facet of the tetrahedron. The internal organization of TET peptidases revealed a highly self-compartmentalized system comprising a crossing network of four access channels extended by vast catalytic chambers in which three active sites are arranged in a circular fashion (19,20). This spatial arrangement is structurally different from all other self-compartmentalized protease complexes of known three-dimensional structure.…”

“…The quaternary structure of archaeal, bacterial, and eukaryal TET assemblies is highly conserved. Their tertiary structures show that they all exhibit a two-domain architecture consisting of a catalytic and a dimerization domain (19,24,25).…”

“…These complexes are built up in an identical fashion and have comparable dimensions. PhTET2 can be defined as a leucyl-aminopeptidase that displays a preference for neutral and aliphatic substrates; PhTET3 is a lysyl-aminopeptidase that hydrolyzes preferentially basic residues, and PhTET1 is a glutamyl-aminopeptidase that shows high specificity toward acidic residues (19,27,28). The comparison of the surface electrostatic potential features of the proteolytic chambers and of the structures of the active site pockets suggest a mechanism of substrate (N-terminal amino acid) discrimination based on the PhTET internal surface electrostatic potential features (19,28).…”

SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs

“…Two approaches are described here to obtain an ab initio structure: randomconical tilt (RCT) and common lines. When an initial 3D model exists, reference-based alignment [10][11][12] can be used.…”