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

Abstract

TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites.

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

“…In addition to their catalytic roles, the metal ions could also control the TETaminopeptidase oligomerization. Such a structural role has been reported for three M42 aminopeptidases: PhTET2 and PhTET3 from P. horikoshii (13,33,34), and PfTET3 from P. furiosus (24). Under chelating conditions, dodecamers disassemble into either dimers (PhTET3) or monomers (PhTET2 and PfTET3).…”

“…The TmPep105012-mer quaternary structure consists of twelve subunits adopting a tetrahedron-shaped architecture (see Figure 1.A) like other available structures of M42 aminopeptidases (13,17,18,24,26,27). The tetrahedron-shaped architecture is often described as the self-assembly of six dimers in such a manner that a dimer lies on each tetrahedron edge.…”

“…The TmPep105012-mer dimerization domain adopts the typical fold of a PDZ-like domain (13). In all structurally characterized M42 aminopeptidases, the 4 helix of the PDZ-like domain is highly flexible (13,15,17,18,24,26,27). The 4 helix, however, was modelled in TmPep105012-mer structure, probably due to its stabilisation in the crystal.…”

“…The M18 family is widely distributed in all domains of life (14) while the M42 family is unique to prokaryotes (23). Several structures of archaeal M42 aminopeptidases have been studied (13,15,17,18,(24)(25)(26) but only one structure has been reported for bacteria (27). Franzetti et al (2002) described the first TET-aminopeptidase structure of the Haloarcula marismortui M42 aminopeptidase, consisting of twelve subunits adopting a tetrahedron-shaped quaternary structure (25).…”

“…Under chelating conditions, dodecamers disassemble into either dimers (PhTET3) or monomers (PhTET2 and PfTET3). Colombo et al (2016) inferred the role of the M1 and M2 sites in the oligomerization: the presence of a metal ion in the M2 site is required for the oligomerization while the M1 site controls protein flexibility (24). According to Macek et al (2017), the dissociation is reversible as dodecamers are formed through random association of dimers (33).…”