Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

Abstract

The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme.

Unraveling self-assembly pathways of the 468-kDa proteolytic machine TET2

“…S5) compatible with dodecameric topology, pointing toward the simultaneous occurrence of multiple assembly pathways. These results are corroborated with an analysis of TET2/TET3 heterododecamer using small-angle neutron scattering, where both the productive assembly octamer and horseshoe hexamers were detected ( 28 ). The apparent discrepancy between the studies using the native and the mutant TET2 sequences suggests that the mutation introduced at the trimerization interface may have substantially altered self-organization.…”

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).…”

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

“…In addition, we have recently demonstrated that the TET peptidase oligomerization process is not random and involves the formation of hexameric intermediates (Appolaire et al, 2013). In the case of the in vitro reconstructed PhTET2:PhTET3 dodecameric particles, the use of SANS and contrast variation revealed that this controlled oligomerization process results in the obligatory formation of catalytic chambers made of the combination of the two different active sites (Appolaire et al, 2014). Thus, after cleavage, the free N-terminus of the polypeptidic product can be easily redirected towards the most suitable active site with respect to the identity of its P1 amino acid.…”

“…Nevertheless, the stoichiometry of one sample could be assessed based on the properties of in vitro reconstituted hetero-oligomers that were produced in order to study the TET oligomerization process. In these studies, the hetero-oligomeric TET particles containing PhTET2 and PhTET3 were obtained by mixing the dimeric precursors recovered after EDTA and basic pH treatment in a re-assembling buffer containing cobalt (Appolaire et al, 2014). The different heterooligomeric forms were purified to homogeneity in enough amounts to be analysed by small angle neutron scattering (SANS).…”

“…The different heterooligomeric forms were purified to homogeneity in enough amounts to be analysed by small angle neutron scattering (SANS). This technique allowed the characterization of a particle composed of six subunits of PhTET2 and six subunits of PhTET3 (Appolaire et al, 2014). Analysed on the Mono Q column, this purified complex eluted at the very same conductivity (35.9 mS cm −1 ) that one of the assemblies purified to homogeneity after coexpression in E. coli (see Fig.…”