# The Role of a Conserved Arg-Asp Pair in the Structure and Function of Tetanus Neurotoxin

## TL;DR

This study explores how a specific pair of amino acids in tetanus neurotoxin affects its structure and function, particularly during membrane insertion and toxin activity.

## Contribution

The study identifies a conserved Arg-Asp pair critical for the structural and functional dynamics of tetanus neurotoxin during membrane insertion.

## Key findings

- Disruption of the Asp-Arg pair in HCT affects thermal stability but not secondary structure.
- Mutations at R711 or D821 alter membrane interaction pH thresholds.
- The D821N mutation increases LC activity in neurons when present in LHNT but not the holotoxin.

## Abstract

Tetanus, a severe and life-threatening illness caused by Clostridium tetani, produces symptoms such as muscle spasms, muscle stiffness and seizures caused by the production of tetanus neurotoxin (TeNT). TeNT causes spastic paralysis through the inhibition of neurotransmission in spinal inhibitory interneurons. This is achieved, in part, through pH-triggered membrane insertion of the translocation (HCT) domain, which delivers the catalytic light-chain (LC) domain to the cytosol. While the function of HCT is well defined, the mechanism by which it accomplishes this task is largely unknown. Based on the crystal structure of tetanus neurotoxin, we identified potential polar interactions between arginine 711, tryptophan 715 and aspartate 821 that appear to be evolutionarily conserved across the clostridial neurotoxin family. We show that the disruption of the Asp-Arg pair in a beltless HCT variant (bHCT) results in changes in thermal stability without significant alterations to the overall secondary structure. ANS (1-anilino-8-napthalene sulfonate) binding studies, in conjunction with liposome permeabilization assays, demonstrate that mutations at R711 or D821 trigger interactions with the membrane at higher pH values compared to wildtype bHCT. Interestingly, we show that the introduction of the D821N mutation into LHNT (LC-HCT only), but not the holotoxin, resulted in the increased cleavage of VAMP 2 in cortical neurons relative to the wildtype protein. This suggests that, as observed for botulinum toxin A, the receptor-binding domain is not necessary for LC translocation but rather helps determine the pH threshold of membrane insertion. The mutation of W715 did not result in detectable changes in the activity of either bHCT or the holotoxin, suggesting that it plays only a minor role in stabilizing the structure of the toxin. We conclude that the protonation of D821 at low pH disrupts interactions with R711 and W715, helping to drive the conformational refolding of HCT needed for membrane insertion and the subsequent translocation of the LC.