Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors

Lyukmanova, Ekaterina N.; Shulepko, Mikhail A.; Шенкарев, Захар О.; Kasheverov, Igor E.; Chugunov, Anton O.; Kulbatskii, Dmitrii S.; Myshkin, Mikhail Yu.; Utkin, Yuri N.; Efremov, Roman G.; Tsetlin, Victor I.; Arseniev, Alexander S.; Kirpichnikov, Mikhaǐl P.; Долгих, Д. А.

doi:10.1016/j.toxicon.2016.06.012

Cited by 21 publications

(24 citation statements)

References 25 publications

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“…Positively charged and aromatic residues are required for interaction of snake three-finger α-neurotoxins with the orthosteric ligand-binding site of nAChRs [ 61 ]. Loop II of WTX, the major determinant of its interaction with the muscle and α7 neuronal receptors [ 62 ], contains three positively charged residues (Arg31, Arg32, and Arg37, Figure 4 H). Similarly, the loops II and III of NTII, essential for interaction with muscle-type nAChRs, also contain several cationic groups (Lys26, Arg32, Lys44, and Lys46, Figure 4 I).…”

Section: Discussionmentioning

confidence: 99%

“…The larger mobility of the NTII loop III is compensated by larger mobility of the WTX loop I ( Figure 3 B). Despite this similarity, the affinity of toxins for muscle-type nAChRs is significantly different (IC 50 ~3 µM and 30 nM for WTX and NTII, respectively) [ 62 , 72 ], indicating the involvement of structural factors other than intramolecular mobility. Nevertheless, the above calculations indicate that the change in conformational entropy of interacting molecules upon the complex formation cannot be neglected.…”

Section: Discussionmentioning

confidence: 99%

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Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors

Paramonov

Kocharovskaya

Tsarev

et al. 2020

IJMS

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Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized β-structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake α-neurotoxins WTX and NTII. Two different topologies of the β-structure were revealed: one large antiparallel β-sheet in Lypd6 and Lypd6b, and two β-sheets in other proteins. α-Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the β-structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors

Paramonov

Kocharovskaya

Tsarev

et al. 2020

IJMS

Self Cite

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“…Escherichia coli has been the work-horse of gene expression for many years, but unfortunately, many recombinant toxins have been expressed as inclusion bodies [14,52,53,54,55], requiring exhaustive in vitro refolding steps to access their biological activities.…”

Section: Discussionmentioning

confidence: 99%

“…Snake toxins have also been expressed into E. coli and this approach has allowed the characterization of toxins with anti-thrombotic [13], anticancer [14], anti-inflammatory [15], antimicrobial activities [16] as well as fused toxins for the development of serotherapy against envenomation [17]. However, when a recombinant protein is synthesized in E. coli , the microenvironment is different from that of the original source, which may lead to protein aggregation, known as inclusion bodies [18].…”

Section: Introductionmentioning

confidence: 99%

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Shimokawa-Falcão

Caporrino

Barbaro

et al. 2017

Toxins

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Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

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“…Similarly, WTX residues Arg‐31 and Arg‐32 were found to contribute to interaction with either of these two receptor subtypes (Lyukmanova et al . ). However, some of the residues within this mandatory set in α‐cobratoxin are more specific for muscle‐type (Lys‐23 and Lys‐49) than for α7 nAChRs recognition (Ala‐28, Lys‐35, and Cys‐26‐Cys‐30) (Antil‐Delbeke et al .…”

Section: Fts Affecting the Cholinergic Systemmentioning

confidence: 97%

The three‐finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions

Kessler

Marchot

Silva

et al. 2017

Journal of Neurochemistry

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Three-finger fold toxins are miniproteins frequently found in Elapidae snake venoms. This fold is characterized by three distinct loops rich in b-strands and emerging from a dense, globular core reticulated by four highly conserved disulfide bridges. The number and diversity of receptors, channels, and enzymes identified as targets of three-finger fold toxins is increasing continuously. Such manifold diversity highlights the specific adaptability of this fold for generating pleiotropic functions. Although this toxin superfamily disturbs many biological functions by interacting with a large diversity of molecular targets, the most significant target is the cholinergic system. By blocking the activity of the nicotinic and muscarinic acetylcholine receptors or by inhibiting the enzyme acetylcholinesterase, three-finger fold toxins interfere most drastically with neuromuscular junction functioning. Several of these toxins have become powerful pharmacological tools for studying the function and structure of their molecular targets. Most importantly, since dysfunction of these receptors/enzyme is involved in many diseases, exploiting the three-finger scaffold to create novel, highly specific therapeutic agents may represent a major future endeavor.

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Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors

Cited by 21 publications

References 25 publications

Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors

Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

The three‐finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions

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