Insights into U-OMP19s structure, a Brucella abortus broad spectrum protease inhibitor (Póster)

SEBASTIÁN KLINKE; KARINA A. PASQUEVICH; MARÍA L. CERUTTI; JULIANA CASSATARO; MARÍA L. DARRIBA; RODOLFO RASIA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

SAIC, SAIB, SAI, SAA, SAB, SAB, SAFE, SAFIS, SAH y SAP

U-Omp19 is a Brucella abortus proteaseinhibitor with immune adjuvant properties. U-Omp19 inhibits the maingastrointestinal proteases (alpha-chymotrypsin, trypsin, pancreatic elastaseand pepsin) and lysosomal cysteine proteases (cathepsin L, B and C). These activitiesmay play a role in UOmp19s adjuvant activity by increasing the half-life ofco-delivered antigens and in B. abortus virulence by protecting it from theaction of host proteases during oral infection establishment. The molecularmechanism and U-Omp19s regions that interact with proteases are still unknown.In this work, we aimed to obtain structural information of U-Omp19 tounderstand deeply its role in virulence and adjuvanticity. In silico analysispredicted that U-Omp19 may belong to I38 family, a family of bacterial proteaseinhibitors characterized by a beta-barrel fold. Based on homology, U-Omp19sinhibitor activity may be among residues 61-158. Secondary structure predictionsuggests 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 andFar-UV CD spectra indicated a high predominance of beta-strand secondarystructure. Assignment of protein resonances in NMR studies using uniformly doublelabeled (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 beta-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. To elucidate U-Omp19s C-terminal function, weobtained a recombinant truncated version (residues 60-158). Far-UV CD spectra confirmedthat it retains the beta-barrel folding, however the inhibitor activity againstalpha-chymotrypsin, trypsin and elastase was lost, indicating that U-Omp19needs at least some part of its N-terminal disordered region for its inhibitoryactivity.