ToxT promoter recognition by ToxR transcription factor, a co-activator within the Vibrio cholerae virulence cascade

Abstract

[eng] Cholera is an acute diarrheal infection caused by the bacterium Vibrio cholerae. An estimated 2.9 million of cases and about 100000 cholera deaths occur annually all over the world. Upon ingestion of the V. cholerae, the bacterium travels to small intestine where it colonizes and produces the cholera toxin. Cholera toxin raises intracellular cyclic AMP and leads to chloride secretion and the subsequent secretory diarrhoea. Cholera toxin production is regulated through the master virulence regulator ToxT. Trascriptional activation of toxT is activated by membrane-localized ToxR, in association with another membrane protein named TcpP. Both ToxR and TcpP work as a two-component regulatory system merged in single proteins: they receive an external signal through its periplasmic C-terminal domain and bind to the toxT promoter by their cytoplasmic N-terminal domains. This project thesis aims at characterizing part of the system by studying ToxR-DNA complexes, since two ToxR molecules are supposed to bind the promoter to recruit TcpP and hence the RNA polymerase for transcription activation. Using X-ray crystallography, we have solved the structure of three complexes of the ToxR DNA binding domain with 20-bp, 40-bp and 25-bp oligonucleotides at 2.0 A, 2.6 A and 3.2 A resolution, respectively. According to the three structures, ToxR is able to bind to an extensive region of the toxT promoter that goes from the position -97 to the position -45. Considering an integrated model of the three structures, there are four ToxR molecules binding the toxT promoter: two molecules bind the DNA in tandem, one molecule binds the ToxR degenerate box and the last one is binding what is supposed to be the TcpP binding site. The structure determination of the three complexes is important to define with more accuracy the ToxR binding site in the toxT promoter. This site is characterized by eleven bases with a high A-T rich region sequence followed by a CATA/CATG/TGTA box, where the last two bases perform direct and specific contacts with the protein. In the three structures, ToxR shows a winged helix-turn-helix (w-HTH) fold. The wing interacts with the minor groove in an A-T rich region sequence while the recognition helix enters in the major groove at the region with a sequence corresponding to a CATA box. We have compared ToxR with the other w-HTH family proteins and we have found a new structural element, the secondary wing, which displays interactions with the DNA. We have analyzed the protein-DNA contacts in the three structures, and also the protein-protein interactions in the ToxR-DNA40 structure, thus validating the data published on ToxR defective mutations. Finally, we put forward a model of toxT promoter activation at molecular level, based on our crystal structures and on what is known in literature and from our collaborators. We propose that ToxR acts as co-activator in the first steps of toxT transcription activation at different levels. First, it would capture the DNA and hold it close to the cytoplasmic membrane, since both ToxR and TcpP are membrane proteins. Second, it would play a key-role in relieving H-NS from the toxT promoter: H-NS binds the DNA and transcription is repressed, but ToxR is able to replace it in a region that goes from the position -97 to the position -45. Third, ToxR would not be recruiting the RNA polymerase directly, but creating the suitable conditions for the action of TcpP. ToxR recruits TcpP probably through a hand-holding mechanism since one of the ToxR binding site is very close to the TcpP's binding site.

[spa] El cólera es una infección diarreica aguda causada por la bacteria Vibrio cholerae. La producción de la toxina colérica se controla a través del regulador maestro de virulencia ToxT, cuya activación se lleva a cabo por las proteínas de membrana ToxR y TcpP. Este proyecto de tesis tiene como objetivo el estudio de los complejos formados por ToxR junto con el ADN, dado que se conoce que ToxR se une al promotor toxT para reclutar TcpP y consecuentemente la ARN polimerasa, produciendo la activación de la transcripción. Mediante cristalografía de rayos X hemos resuelto la estructura de tres complejos del dominio de unión a ADN de ToxR con oligonucleótidos de 20, 40 y 25 pares de bases a resoluciones de 2.0 A, 2.6 A y 3.2 A, respectivamente. De acuerdo con las tres estructuras, ToxR es capaz de unirse a una amplia región del promotor toxT que se expande desde la posición -97 hasta la posición -45. Teniendo en cuenta el modelo integrado de las tres estructuras, cuatro moléculas de ToxR se unen al promotor toxT en tándem e invertidas. En las tres estructuras, ToxR muestra un tipo de plegamiento winged helix-turn-helix (w-HTH). El ala (wing) interactúa con el surco menor del ADN, mientras que la hélice de reconocimiento penetra en el surco mayor. Comparando ToxR con el resto de proteínas de la familia w-HTH, hemos encontrado un nuevo elemento estructural, el ala secundaria (secondary wing), que interacciona con el ADN. La determinación de la estructura de los tres complejos es importante para definir con mayor precisión el sitio de unión de ToxR en el promotor toxT. Este sitio se caracteriza por once bases con una secuencia rica en A-T seguida de una caja CATA/CATG/TGTA, donde las dos últimas bases contactan directamente y específicamente con la proteína. Proponemos que ToxR actuaría como co-activador de la transcripción de toxT a diferentes niveles: (i) podría ser responsable de capturar el ADN y mantenerlo cerca de la membrana citoplasmática, (ii) podría jugar un papel clave en el desplazamiento de H-NS, (iii) podría reclutar TcpP y estabilizar su interacción con el promotor.