Naslov
The first X-ray structure of prokaryotic dipeptidyl peptidase III

Autori
Sabljić, Igor ; Gruber, Karl ; Macheroux, Peter ; Luić, Marija

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Acta Crystallographica Section A Foundations and Advances / - , 2015, S202-s203

Skup
29th European Crystallographic Meeting

Mjesto i datum
Rovinj, Hrvatska, 21.08.2015. - 29.08.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
dipeptidil-peptidaza III ; metalopeptidaze ; porodica M49 ; Bacteroides thetaiotaomicron ; Caldithrix abyssi
(dipeptidyl peptidase III ; metallopeptidase ; M49 family ; Bacteroides thetaiotaomicron ; Caldithrix abyssi)

Sažetak
Dipeptidyl peptidase III (DPP III) is a widely distributed cytosolic zinc peptidase from the M49 family, which hydrolyses dipeptides from the N-termini of its peptide substrates. Until now, most studies on DPP III were done on orthologs from eukaryotic organisms, showing that DPP III participates in the intracellular protein catabolism and oxidative stress response. The knowledge about prokaryotic DPP III is very scarce, particularly data on its catalytic mechanism, physiological function and structural characteristics are lacking. We have been studying two bacterial orthologs: DPP III from the human gut symbiont Bacteroides thetaiotaomicron (Bt) and from Caldithrix abyssi (Ca), which inhabits hydrothermal vents. Both proteins were produced in Escherichia coli and then purified. Diverse crystallization screens were applied and wild-type CaDPP III crystals were obtained. Crystallization experiments with wild-type BtDPP III failed probably due to charge heterogeneity. In order to overcome this problem, specific amino acid replacements (all cysteines were replaced by serines) were introduced and crystals of the variant protein were grown. Datasets for both DPP III proteins were collected at BESY II, Germany. Up to date, all efforts to solve the structures of the two proteins using molecular replacement method and anomalous dispersion of the zinc ions, were unsuccessful. Therefore, selenomethionine labeled BtDPP III, non-cysteine variant, was prepared and the crystals were obtained in the same crystallization condition as the non-labelled protein. Diffraction data up to 1.8 Å resolution were collected at Elettra Trieste, Italy, and the first prokaryotic DPP III structure was solved using single-wavelength anomalous dispersion of the selenium atoms. BtDPP III crystallized in space group P3121 with unit cell dimensions a=103.5 Å and c=141.6 Å and one molecule per asymmetric unit. The overall fold is typical for a member of the M49 family with two domains separated by a wide cleft. The upper domain is mostly helical with a tetracoordinated zinc ion, while the lower domain contains mixed secondary structure elements with a five-stranded β-barrel core. The main structural difference between BtDPP III and eukaryotic DPP III is the absence of a 30 amino acid loop in upper domain. In human DPP III this loop contains the ETGE motif which is supposed to be responsible for binding KEAP1, a repressor protein from the Keap1-Nrf2 signaling pathway.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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