Structural and dynamic insights into the C-terminal extension of cysteine proteinase B from Leishmania amazonensis.

Cysteine proteinase B (CPB) is a significant virulence factor for Leishmania infections. Upon processing from its zymogen form, it happens a release of the immunomodulatory CPB C-terminal extension (cyspep) into the cytoplasm of the macrophage. Epitopes derived from this fragment were shown to influence the proportion of lymphocytes CD8+ upon infection, favoring the parasite escaping from the host́s immune system. At present, there is no available structural data of cyspep, which impairs a proper understanding of its biological functions. Here, we attempted to build molecular models for this fragment and subsequently evaluate their stabilities in aqueous solution from molecular dynamics simulations analysis. Characterization of our models obtained with distinct techniques (comparative modeling, threading, and ab initio) indicates a prevalence of ß-sheets in agreement with consensus secondary structure predictions. Simulation data supported this finding since the formation of new strands, along with a rapid disruption of helical content, were observed. Overall, this study provides a rationalization of epitope mapping data and an improved understanding of cyspep antigenicity.

Proposição de modelos tridimensionais da extensão COOH-terminal da cisteíno-protease B de Leishmania (Leishmania) amazonensis / Proposition of three-dimensional models of the cooh-terminal cysteine protease B extension of leishmania (leishmania)

A Leishmania Leishmania amazonensis é um importante agente etiológico da leishmaniose tegumentar em humanos no Brasil. Este parasito apresenta mecanismos de adaptação que podem ser guiados por suas proteases onde se destacam as cisteíno proteases CP, sendo a CPB a mais estudada dentre as CPs já descritas em Leishmania spp. O foco deste trabalho é a região COOH terminal da CPB cyspep, por suas reconhecidas propriedades modulatórias sobre o sistema imune de camundongos. No presente estudo busca se propor um modelo estrutural da cyspep visando estabelecer sua relação estrutura função. O estudo foi conduzido por abordagem in silico, desenvolvendo o modelo tridimensional através de técnicas de modelagem comparativa, threading e métodos de novo, e analisando a estabilidade estrutural por dinâmica molecular. A associação do uso de servidores online que utilizam as abordagens de modelagem comparativa, threading e métodos de novo para predição tridimensional de proteínas foi favorável à resolução de 72 modelos da cyspep. Através de informações sobre predição de estrutura secundária e ligações dissulfeto, dos modelos gerados para cyspep, 11 foram selecionados para simulações de dinâmica molecular, sendo dois modelos obtidos por modelagem comparativa Comp, Fugue, 5 por threading IT1, M4, M8, L3 e SP5 e 4 por métodos de novo Q5, Q7, D9 e R1. Os 11 modelos foram submetidos a simulação por dinâmica molecular por um tempo de 200 ns. Os modelos Fugue e IT1 também foram submetidos a simulações de dinâmica molecular sob a influência de diferentes campos de força para melhor avaliar seu conteúdo de estrutura secundária. Nossos resultados sugerem que a cyspep pode adotar uma conformação composta principalmente de folhas beta, as quais se mantiveram estáveis após longas simulações de dinâmica molecular sob a influência de diferentes campos de força; enquanto o teor helicoidal foi rapidamente desfeito...

Leishmania (Leishmania) amazonensis is an important etiological agent of cutaneous leishmaniasis in humans, in Brazil. This parasite has adaptive mechanisms that can be modulated by their proteases, characterizing the cysteine protease (CP) as themost extensively studied CPB among the CPs described for Leishmania spp. This work was focused on the COOH-terminal region of CPB (cyspep), recognized for their modulatory properties on the immune system of mice. This present study aims topropose a structural model for the cyspep trying to determine its structure-function relationship. The present study was conducted by in silico to develop a three-dimensional model using comparative modeling, threading and de novo methods, andmolecular dynamics to analyze its structural stability. The association of many online servers using the approaches of comparative modeling, threading and de novo methods for the prediction of the three-dimensional structure of protein was favorablefor the achievement of 72 three-dimensional models for cyspep. Through secondary structure and disulfide bonds prediction, out of the 72 models generated for cyspep, 11 were selected for molecular dynamics simulations, of which 2 models were obtained by comparative modeling (Comp, Fugue), 5 by threading (IT1, M4, M8, L3and SP5) and 4 de novo methods (Q5, Q7, D9 and R1). The eleven models were submitted to molecular dynamics simulations for a computational time of 200 ns. The Fugue and IT1 models were also subjected to molecular dynamics simulations under different force fields to better assess their secondary structure content and determinethe impact of the force field on the structure. Our results suggest that the cyspep might adopt a conformation composed of mostly beta-sheets, which remained stable after long molecular dynamics simulations under the influence of different force fields while the helical content was rapidly disrupted...