Insights into U-Omp19`s structure, a Brucella abortus broad spectrum protease inhibitor.

DARRIBA ML; CERRUTI, MARÍA L.; KLINKE, SEBASTIÁN.; RASIA, RODOLFO; CASSATARO, JULIANA; PASQUEVICH KARINA ALEJANDRA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017; 2017

Sociedades Biocientíficas. Sociedad Argentina de Inmunología.

U-Omp19 is a Brucella abortus protease inhibitor with immune adjuvant properties. U-Omp19 inhibits the main gastrointestinal proteases (α-chymotrypsin, trypsin, pancreatic elastase and pepsin) and lysosomal cysteine proteases (cathepsin L, B and C). These activities may play a role in U Omp19`s adjuvant activity by increasing the half-life of co-delivered antigens and in B. abortus virulence by protecting it from the action of host proteases during oral infection establishment. The molecular mechanism and U-Omp19´s regions that interact with proteases are still unknown. In this work, we aimed to obtain structural information of U-Omp19 to understand deeply its role in virulence and adjuvanticity.In silico analysis predicted that U-Omp19 may belong to I38 family, a family of bacterial protease inhibitors characterized by a beta-barrel fold. Based on homology, U-Omp19´s inhibitor activity may be among residues 61-158. Secondary structure prediction suggests that residues 1-60 are disordered and may not present a regular structure.SLS and DLS studies showed it behaves as a globular monomer of 16,8 kDa and Far-UV CD spectra indicated a high predominance of β-strand secondary structure.Assignment of protein resonances in 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 (residues 70-158). 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, we obtained a recombinant truncated version (residues 60-158). Far-UV CD spectra confirmed that it retains the beta-barrel folding, however the inhibitor activity against α-chymotrypsin, trypsin and elastase was lost, indicating that U-Omp19 needs at least some part of its N-terminal disordered region for its inhibitory activity.