Insights into U-omp19`s structure, a Brucella abortus broad spectrum protease inhibitor

DARRIBA, MARIA LAURA; CERRUTI, MARIA LAURA; RACIA, RODOLFO; CASSATARO JULIANA; PASQUEVICH KARINA ALEJANDRA

Congreso; XLII Congresso da Sociedade Brasileira de Biofísica - Sao Paulo 2017 (SBBf 2017); 2017

U-Omp19 is a Brucellaabortus protease inhibitor withimmune adjuvant properties. U-Omp19 inhibits the main gastrointestinalproteases (α-chymotrypsin,trypsin, pancreatic elastase and pepsin) and lysosomal cysteine proteases(cathepsin L, B and C) [1, 2]. Theseactivities may play a rolein U‑Omp19`s adjuvant activity by increasing the half-life of co-deliveredantigens and in B. abortus virulence by protecting it from the action ofhost proteases during oral infection establishment. The molecular mechanism and U-Omp19´sregions that interact with proteases are still unknown. In this work we aimed to obtain structuralinformation of U-Omp19 to understand deeply its role in virulence andadjuvanticity.In silico analysis predicted that U-Omp19may belong to I38 family, a family of bacterial protease inhibitorscharacterized by a beta-barrel fold.SLS and DLS studies showed it behaves as aglobular monomer of 16.8 kDa and Far-UV CD spectra indicated a highpredominance of β-strand secondary structure.Assignment of protein resonances in NMRstudies using uniformly double labeled (15N, 13C) U-Omp19confirmed that it bears a flexible N-terminal region (residues 1-64) and aC-terminal compact core of eight anti-parallel β-strands (residues 70-158). The lowestenergy structure obtained from fold calculations using backbone chemical shiftsas restraints is similar to the structures of other inhibitors from I38 family.To elucidate U-Omp19´s C-terminalfunction, we obtained a recombinant truncated version (residues 60-158). Far-UVCD spectra confirmed that it retains the beta-barrel folding, however theinhibitor activity against α-chymotrypsin, trypsin and elastase was lost, indicating that U-Omp19 needs at least some partof its N-terminal disordered region for its full inhibitory activity.