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 DNA-binding. 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. Assuming ToxT is similar in mechanism to other AraC proteins, helix six from HTH1 and helix nine from HTH2 become aligned with the help of helix seven (the polar linking region) to allow binding to major consecutive grooves of target DNA.. The conformation of helix seven is dependent on the ligand bound. Ligand-binding. 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. K230 is at the end of helix seven, and binding to K31 causes pulling helix six into an unfavorable conformation that disallows DNA binding. 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 by giving helix seven and thus six freedom, allowing DNA binding. Dimerization. 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]

Further Study

Conclusive results about what activates ToxT itself has not yet been found. The varying activity of ToxT dependent on the presence of cis-palmitoleate or other unsaturated fatty acids represents a detailed method of effective pathogenicity in humans, but may not be a reasonable target for drug treatment. By restricting transcription (and thus translation and protein production) of virulence genes until the bacterium is determined to be in a favorable location for infection, Vibrio cholerae avoids wasting energy producing virulence factors that will just be cleared by the intestine. This is a specific mechanism to ensure that the bacterium also injects CT and TCP where they will do the most damage, resulting in an infection.

Proteopedia Page Contributors and Editors (what is this?)

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