StIIR124C: un nuevo mutante para la caracterización del mecanismo de formación de poros de Sticholisina II en células / STIIR124C: a new mutant to characterize the mechanism of pore formation by Sticholysin II in cells

La Sticholisina II (StII) es una actinoporina producida por la anemona de mar Stichodactyla helianthus (Anthozoa: Stichodactylidae). Su estructura tridi-mensional se caracteriza por un nucleo central de hojas-β intercaladas flanqueado por dos helices-α, un sitio de union interfacial y lazos que interconectan las fibras β. El mecanismo de formacion de poros de las actinoporinas transcurre mediante varias etapas dentro la que se encuentra: la union a la membrana, la oligomerizacion, el despliegue del extremo amino y finalmente la formacion del poro. Dicho mecanismo no ha sido completamente dilucidado, fundamentalmente las bases moleculares de la etapa de oligomerizacion. En general, las actinoporinas no poseen residuos de cisteinas en su estructura primaria. La obtencion de mutantes de cisteina permite el marcaje con numerosas sondas fluorescentes o de espin especificas a los grupos sulfidrilos (SH) para el estudio de la interaccion proteina-membrana. Esto impone la necesidad de obtener variantes mutadas de la proteina que incorporen la cisteina a su estructura. En este trabajo, se obtuvo el mutante StII R124C en el que se sustituyo el aminoacido Arg124, localizado hacia el extremo carboxilo de StII, por un residuo de cisteina. StII R124C se expreso en el sistema de Escherichia coli y se purifico en un solo paso mediante cromatografia de intercambio cationico. La actividad biologica de StII R124C evaluada mediante la lisis de celulas eritrocitarias no mostro variacion, lo que sugiere que la sustitucion de Arg124 por cisteina en StII recombinante no afecta la capacidad formadora de poros de la proteina. Asi, el mutante StII R124C constituye una alternativa para la realizacion de estudios de relacion estructura-funcion. Palabras clave: citolisinas, proteina formadora de poros, mutantes de Cys, actividad hemolitica ABSTRACT Sticholysin II (StII) is an actinoporin produced by the sea anemone Stichodactyla helianthus (Anthozoa: Stichodactylidae). The tridimensional structure of actinoporins display a common fold characterized by a β-sandwich core flanked by two α-helices, a membrane recognition site and loops interconnecting all β-sheets. Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, the molecular details of the mechanism of oligomerization are not clear. Actinoporins do not have cysteine residues in their primary structure. Cysteine mutants allow the labeling of the proteins with different dyes as fluorescent or spin probes specifically to sulfhydryl groups (SH). They can be used for the study of the interaction of these proteins with membrane. This imposes the need to obtain mutated variants of the protein that incorporate cysteine into its structure. In this work, we obtained a mutant of StII in which the residue Arg124, located near the C-terminus, was replaced by cysteine (StII R124C). StII R124C was ex-pressed in a bacterial Escherichia coli system and purified in a single step by cation exchange chromatography. StII R124C mutant showed the same pore-forming activity as the native protein suggesting that the substitution of Arg124 for cysteine in recombinant StII does not affect the pore-forming capacity of the protein. Thus, the mutant StII R124C constitutes an alternative for the performance of studies of structure-function relationship. Keywords: cytolysins, pore-forming protein, Cys mutants, hemolytic activity