INSIGHTS INTO U-OMP19`S STRUCTURE, A Brucella abortus BROAD SPECTRUM PROTEASE INHIBITOR.

DARRIBA, MARIA LAURA; CERRUTI, MARIA LAURA; KLINKE S; RASIA, RODOLFO; CASSATARO, JULIANA; PASQUEVICH KARINA ALEJANDRA

Congreso; Reunion Conjunta de Sociedades de Biociencias 2017; 2017

U-Omp19 is a Brucella abortus protease inhibitor with immune adjuvant properties. U-Omp19inhibits the main gastrointestinal proteases (α-chymotrypsin, trypsin,pancreatic elastase and pepsin) and lysosomal cysteine proteases (cathepsin L,B and C). These activities mayplay a role in U‑Omp19`s adjuvant activity by increasing the half-life ofco-delivered antigens and in B. abortus virulence by protecting it fromthe action of host proteases during oral infection establishment. The molecular mechanism andU-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 itsrole in virulence and adjuvanticity.Insilico analysis predicted that U-Omp19 may belong to I38 family, a family ofbacterial protease inhibitors characterized by a beta-barrel fold. Based onhomology, U-Omp19´s inhibitor activity may be among residues 61-158. Secondarystructure prediction suggests that residues 1-60 are disordered and may notpresent a regular structure.SLSand DLS studies showed it behaves as a globular monomer of 16,8 kDa and Far-UVCD spectra indicated a high predominance of β-strand secondary structure.Assignmentof 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 calculationsusing backbone chemical shifts as restraints is similar to the structures ofother inhibitors from I38 family.Toelucidate U-Omp19´s C-terminal function, we obtained a recombinant truncatedversion (residues 60-158). Far-UV CD spectra confirmed that it retains thebeta-barrel folding, however the inhibitor 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 inhibitory activity.