Clostridium tetani Tetanus Neurotoxin (TeNT)

Anu Muppirala '19 and Haofan Li '20

Contents:

I. Introduction




Tetanus neurotoxin (TeNT) is a potent and possibly lethal substrate produced by the anaerobic bacteria Clostridium tetani [1]. A member of the clostridial neurotoxin [2] (CNT) family, TeNT is the causative agent of Tetanus: a disease associated with mortality rates ranging from virtually 0% in the United States to nearly 80% in other regions of the world with limited access to health care and adequate medical treatment [3].

TeNT is also known as spasmogenic toxin, reflective of the toxin�s effects on skeletal muscle function. Mechanistically, upon entry into the bloodstream, TeNT targets neuromuscular junctions, where it binds to peripheral motor neurons. Subsequently, the toxin is endocytosed, and transported in a retrograde fashion to reach the presynaptic interneurons at the spinal cord. Here, TeNT enters the interneuron terminal where it employs its zinc-dependent proteinase (metallaprotease) domain to directly cleave the synaptic vesicle associated protein, synaptobrevin [4]. As a member of the SNARE [5] (Soluble NSF Attachment Protein Receptor) complex, synaptobrevin is critical to neurotransmitter release from the vesicle via mediating lipid bilayer fusion of the vesicle membrane with the presynaptic terminal membrane of the interneuron.

These motor interneurons are responsible for balancing skeletal motor output by downregulating muscle contraction through inhibitory GABA [6] and glycine[7] neurotransmitter release. By inhibiting the inhibitory interneuron circuit, TeNT results in hyperactive or spastic skeletal motor movement, a hallmark of Tetanus.

wild

Figure 1A. Schematic of normal interneuron signaling. Presynaptic interneuron releases inhibitory neurotransmitter from synaptic vesicles, which subsequently inhibit the postsynaptic neuron from signaling.

 inhabit

Figure 1B. Schematic of TeNT disruption of interneuron signaling. Synaptobrevin and SNARE complex in presynaptic interneuron inhibited, therefore, no inhibitory neurotransmitter is released from synaptic vesicles. Subsequently, the postsynaptic neuron is no longer inhibited, leading to hyperactivity and spastic motor movement.

II. General Structure




is a 150 kDa polypeptide that is cleaved post-translationally and forms a dimer to yield an active structure by host or bacterial proteases. Within one unit of the homodimer, two domains, the light chain and the heavy chain , are connected by a single disulfide bridge . Both the LC and HC structural elements are unique and carry out distinct functions.

The domain of the LC contains a 50-kDa zinc-dependent metalloproteinase. The zinc cation interacts with residues H233, E234, and H237 of the . This active zinc protease cleaves synaptobrevin, inhibiting vesicle fusion with the terminal plasma membrane and subsequent neurotransmission. On the other hand, the , residues , are surrounded and stabilized by the .

The is composed of two 50-kDa domains, the HN translocation domain and the HC binding domain. Upon binding the neuronal membrane, the HC changes its conformation, thus altering the translocation domain. The HN then assists the LC to translocate through the neuronal plasma membrane into the terminal. Here, the catalytic activity of the LC can access and cleave synaptobrevin.

The HC of TeNT binds the toxin to the presynaptic interneuron membrane and forms additional intramolecular interactions to stabilize the binding. For example, a few key interactions between the HC and HN occur, including the formed by S640 and H936, the formed by R802 and D940, and between K865 and three residues (S900, T885, and K882).

In coninuation, the HC interacts with the LC through hydrogen bonding of the backbone. Residues (NSSVITY) comprise a beta-sheet of the HC , which interacts with an opposing beta-sheet of the LC formed from amino acid residues (NITSLT) . The hydrophobic residues facilitate a favorable position for HC and LC interaction and for overall stability of the TeNT protein.

Additionally, there are secondary interactions between the residues (KSEY)of the LC and the residues (NNDI) of the HC that contribute to stability as well. Other than the N893 and S410 residues, which form a bond through the backbone, all the other combinations, (S409 and D894, N893 and Y411 ), engage in ionic bonding between the side chains[8].

Video 1. The C terminal domain of the heavy chain is translocated to assist TeNT binding to membrane.[8]

III. Molecular interaction




While TeNT's ability to bind and enter the presynaptic interneuron membrane is defined, specific membrane components that interect with TeNT are currently under investigation. One example, GM1a, is a sialic acid-containing glycosphingolipid that is found in normal cell membranes and comprises about 5% of membrane lipids. Interestingly, GM1a is particularly concentrated in neuronal membranes, where it carries out neuroprotective functions such as stimulating neurotrophin release, and aiding in neuron health, function, and signaling [9]. Since GM1a is implicated specifically in regulating neurotransmission, inhibition by TeNT may contribute to dysregulated motor neuron signaling and spastic skeletal muscle movement

Several hydrogen bonds contribute to the interaction between the GM1a molecule and atoms of the TeNT amino acid residues, which are listed in the table below.


Protein atom Distance(A) GM1a atom Button
N1219 2.8 GalNAc3(O4)
N1219 2.9 GalNAc3(O5)
D1222 3.3 Gal4(O6)
D1222 2.7 GalNAc3(O4)
H1271 3.0 Gal4(O4)
H1271 3.0 Gal4(O5)
H1271 3.3 Gal4(O6)
W1289 3.3 Sia6(O1B)

Table 1. TeNT contains two ganglioside binding sites. Listed are various hydrogen bond interactions made in the GM1a-specific binding pocket between TeNT amino acid residues and the GM1a ganglioside. The two gangliosides that TeNT binds differ merely by a single N-acetylneuraminic acid group, which is present in the other ganglioside, GD1a but absent in GM1a. However, both gangliosides are involved in the same hydrogen bond interactions. Most noteably, the conserved tryptophan 1289 residue is key to GM1a recognition through a with Gal4, which is stabilized by hydrophobic interactions with tyrosine 1290. [8]




TeNT undergoes a pH-mediated conformational change resulting from the proton pumping activity of ATPases in the endosomal/vesicle membrane. This pH dependency is critical to regulating TeNT's conformation to allow it to be successfully transported to the interneuron and inhibit vesicle release. Experimental data reveal that TeNT exhibits a "compact conformation" when the pH is lower than 5.5. However, TeNT enters an "extended conformation" when the pH reaches higher than 6.5. The physiologically relevant change in the maximum intramolecular distance of TeNT between these pH states ranges from 125 A at pH 5.0 to approximately 140 A at pH 8.0.

In context of its function in neurons, TeNT exists in its extended conformation during retrograde axonal transport, where the environment is a neutral pH. As it encounters the acidic environment of synaptic vesicles near the presynaptic interneuronal membrane, TeNT enters the compact conformation, where the HC assists the LC in order for TeNT to travel across the cell membrane [8].

pHchange

Figure 2. Structure of extended (ph 8, left) and compact (ph 5, right) conformational states of TeNT, including change in intramolecular distance.[8]

Video 2. TeNT exhibits pH-dependent dynamism. Extended and compact conformational states define TeNT in basic and acidic pH microenvironments respectively. [8].

V. References

[1] Clostridium tetani - microbewiki. (n.d.). Retrieved December 16, 2018

[2] Goonetilleke, A., and Harris, J. (2004). CLOSTRIDIAL NEUROTOXINS. Journal of Neurology, Neurosurgery, and Psychiatry, 75(Suppl 3), iii35�iii39.

[3] Thwaites, C. L., and Loan, H. T. (2015). Eradication of tetanus. British Medical Bulletin, 116(1), 69�77.

[4] Synaptobrevin. (2018). In Wikipedia. Retrieved 02:19, December 18, 2018.

[5] SNARE (protein). (2018). In Wikipedia. Retrieved 02:20, December 18, 2018.

[6] Gamma-Aminobutyric acid. (2018). Wikipedia. Retrieved 02:20, December 18, 2018.

[7] Glycine. (2018). In Wikipedia. Retrieved 02:21, December 18, 2018.

[8] Masuyer G., Conrad J., and Stenmark P. (2017). The structure of the tetanus toxin reveals pH-mediated domain dynamics. EMBO reports. Vol. 18, No. 8, pp. 1306�1317, 2017.

[9] Xi, A. P., Xu, Z. X., Liu, F. L., and Xu, Y. L. (2015). Neuroprotective effects of monosialotetrahexosylganglioside. Neural regeneration research, 10(8), 1343-4.

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