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.
Highlights
Cytosolic proteolysis is a key cellular process
The quaternary structures of PhTET1 described in this paper reveal a novel type of elaborated self-compartmentalized orga
The tetrahedral organization of PhTET1-12s indicates a function as a peptide scavenger, the geometry and electrostatic charge repartition in the channels being involved in the substrate peptide binding and orientation toward the catalytic chambers
Summary
PhTET2, P. horikoshii TET2; HmTET, Haloarcula marismortui TET; PhTET1-12s, P. horikoshii TET1 12-subunit complex; PhTET1-24s, P. horikoshii TET1 24-subunit complex; PIPES, piperazine-N,NЈbis(2-ethanesulfonic acid); pNA, 4-nitroaniline; AMC, 7-amino-4-methylcoumarin; AAP, V. (formerly Aeromonas) proteolyticus aminopeptidase; HPLC, high pressure liquid chromatography. Peptidase Tetrahedral and Octahedral Assemblies displays a nonprocessive catalytic activity against N-terminal aliphatic and neutral amino acids (18). The structure of the PhTET2 edifice reveals the self-compartmentalizing nature of the complex (19, 20). In the genome of P. horikoshii, the open reading frame PH0519 encodes a homolog of PhTET2 that has been characterized as a cobalt-activated peptidase, suspected to be a deblocking aminopeptidase and thereby named DAP (21). The three-dimensional structures of PH0519, presented here, show that it can assemble alternatively as a tetrahedral dodecameric particle ( called PhTET1-12s) or as an octahedral tetracosameric edifice (PhTET1-24s). These assemblies are different from those observed in the other self-compartmentalized protease complexes of known three-dimensional structures. A mechanism of action for PhTET1 based on its peculiar compartmentalization and structural features is proposed
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