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

DARRIBA, M.L; CASSATARO, J.; RASIA, R; CERUTTI, M.L; PASQUEVICH, K.A.

Santos

Congreso; XLII Brazilian biophysical society congress; 2017

Brazilian biophysical society

U-Omp19 is a Brucellaabortus protease inhibitor withimmune adjuvant properties. U-Omp19 inhibits the main gastrointestinalproteases (α-chymotrypsin, trypsin, pancreatic elastase and pepsin) andlysosomal cysteine proteases (cathepsin L, B and C) [1, 2]. These activities may play a role in U‑Omp19`s adjuvant activity by increasing thehalf-life of co-delivered antigens and in B. abortus virulence byprotecting it from the action of host proteases during oral infection establishment. The molecular mechanism and U-Omp19´s regionsthat 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-Omp19 may belong to I38 family, a family of bacterial protease inhibitors characterizedby 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 resonancesin NMR studies using uniformly double labeled (15N, 13C) U-Omp19 confirmed that it bears a flexible N-terminal region (residues 1-64) and a C-terminal compact core of eight anti-parallel β-strands (residues70-158). The lowest energy structure obtained from fold calculations using backbone chemical shifts as restraints is similar to the structures of other inhibitors from I38 family. To elucidate U-Omp19´s C-terminal function, weobtained a recombinant truncated version (residues 60-158). Far-UV CD spectra confirmed that it retains the beta-barrel folding, however the inhibitoractivity 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.References:1.Ibanez,A.E., et al., A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion whiletriggering specific mucosal immune responses. J Control Release, 2015. 220(PtA): p. 18-28.2.Coria, L.M., et al., A Brucella spp. Protease Inhibitor Limits Antigen Lysosomal Proteolysis, Increases Cross-Presentation, and Enhances CD8+T Cell Responses. J Immunol, 2016. 196(10): p. 4014-29.