Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/β subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for β-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin β-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: an example of snake venom phospholipases A2 versatility

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.