Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones.Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by E. coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per litre of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner.Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

Human glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) is an established marker for prostate-cancer diagnosis as well as a candidate therapeutic target for the treatment of diverse pathologies that involve glutamatergic transmission. Structural data on GCPII are thus valuable for the design and optimization of GCPII-specific inhibitors and diagnostic probes. The currently available structure of ligand-free GCPII was refined to a resolution of 3.5 A. This work reports the structure of the protein refined to 1.65 A resolution, with crystallographic values of R = 0.207 and R(free) = 0.228. The new structure extends the resolution appreciably and the new model based on this data shows significant differences when compared with the previously published model.

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII, Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (FolGlun), which are the provitamin form of folic acid (also known as vitamin B9). Despite the role of GCPII as a folate hydrolase, nothing has been known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In this study we present 1) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates, 2) X-ray structure of the His475Tyr variant at 1.83 Å resolution, 3) study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu, and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates and 4) comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild type and His475Tyr variant are functionally identical.

We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.