The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero‐subunit peptidasome with enhanced peptide destruction properties

Abstract

SummaryTET aminopeptidases assemble as large homododecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.

Structure of the dodecamer of the aminopeptidase APDkam598 from the archaeonDesulfurococcus kamchatkensis

“…It may be speculated that these Trp residues can participate, for example, in protein-protein interactions. It was recently found that PhTET2 and PhTET3, which have the highest sequence similarity to APDkam589 among the known structures, can assemble to form heterooligomeric complexes (Appolaire et al, 2014). Further investigations are needed to verify the possible role of Trp residues in the functions of APDkam589.…”

“…Another interesting line of research is the investigation of the selfoligomerization of the 12-subunit complex (Appolaire et al, 2013). Recently, it has been reported that PhTET2 and PhTET3 assemble to form heteromeric complexes (Appolaire et al, 2014).…”

How metal cofactors drive dimer–dodecamer transition of the M42 aminopeptidase TmPep1050 of Thermotoga maritima

“…In Pyrococcus horikoshii, four TET-aminopeptidases -PhTET1, PhTET2, PhTET3, and PhTET4 -have been described and each of them displays a different substrate specificityaspartyl-, leucyl-, lysyl-, and glycyl-aminopeptidase activity, respectively (15)(16)(17)(18). Remarkably, heterocomplexes, made of PhTET2 and PhTET3, have been reported, leading to the assumption of the peptidasome particle existence (19,20).…”

Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites

“…The oligomerization interface is supported by both the proteolytic domain and the PDZ-like domain, driving the association of the dimeric building blocks into the dodecameric assembly 15 16 . It has been recently shown that TET dimers are present in vivo 17 and that their activity against long peptides is considerably lower compared to the dodecameric particle 16 , suggesting a fine regulation of TET activity by controlling its oligomerization.…”