Abstract
Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide- and linear-linked SAMP1-MoaE in purified form. Cleavage of linear linked SAMP1-MoaE was dependent on the presence of the SAMP domain and the C-terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl2 . HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active-site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin-like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of 'free' SAMP available for protein modification. HvJAMM1-type proteins are thought to release the SAMPs from proteins modified post-translationally as well as those synthesized as domain fusions.