ToxT

The crystal structure of ToxT is resolved in monomeric form, after isolation from Vibrio cholerae strain O395.

Introduction

ToxT is a molecule at the end of a transcriptional cascade that autoregulates the transcription of the primary virulence factors of Vibrio cholerae[1] and itself. These two factors, cholera toxin (CT)[2] and the toxin co-regulated pilus (TCP), are instrumental in causing the disease cholera[3]. This is an intestinal infection resulting in massive water loss in the affected individual, causing extreme dehydration.[4]

spinqualitylabelsall models ToxT, 1.9 Angstrom resolution crystal structure Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Ligand In this resolved structure, cis-palmitoleate[1] is present and bound in the beta sheet barrel (further discussed below). This unsaturated fatty acid reduces virulence expression in Vibrio cholerae. Other Structural Features ToxT belongs to a family of transcriptional regulators headed by and known as AraC. The AraC family is characterized by a 100 amino acid region of sequence similarity that forms a DNA-binding domain with two helix-turn-helix motifs (helix in blue, turn in teal). The two HTH regions are linked by a very polar alpha helix (shown in black). The overall domain is composed of seven alpha helices, and is located at the C-terminus. A nine-stranded beta sheet sandwich or "jelly-roll" with three other alpha helices (overall making up the N-terminus) contain a binding pocket. This is largely made up of residues from the N-terminus (Y12, Y20, F22, L25, I27, K31, F33, L61, F69, L71, V81, and V83), and a few from the C-terminus (I226, K230, M259, V261, Y266, and M269). The pocket is highly hydrophobic, and has a known volume of 780.9 Angstroms. This pocket contains a ligand: cis-palmitoleate which appears to have a negative effect on virulence when present in vitro. The cis-palmitoleate forms salt bridges with residues K31 and K230. This unsaturated fatty acid, like other UFAs,[2] tend to inhibit genes under the control of ToxT. The Specifically, the cis-palmitoleate appears to bind directly to ToxT, change its conformation, and thus lower the ability to bind DNA and form dimers. The presence of UFAs is associated with being in the lumen of the intestine during the bacterial infection. Here, binding of PAM brings K31 and K230 together and essentially closes off ToxT. In lower concentration of fatty acids, ie: after penetrating the intestine's mucus, the two lysine residues repel each other and give ToxT an open conformation, allowing it to bind DNA. Though the structure shown is a monomer with two overall domains (N-terminal and C-terminal), ToxT tends to form a dimer. The preferred state of ToxT varies between promoters, but binding to the ctx promoter to generate cholera toxin appears to be possible only in the dimer form.ToxT binds to thirteen base pair sequences (can be single, direct, or inverted repeats) called toxboxes in order to activate their respective promoters.[3]

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Ingrid Youngworth, Yang Yang, Michal Harel, Jaime Prilusky, Alexander Berchansky