Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling

Dubovskii, Peter V.; Dubova, K. M.; Bourenkov, Gleb; Starkov, Vladislav G.; Konshina, Anastasia G.; Efremov, Roman G.; Utkin, Yuri N.; Samygina, V.R.

doi:10.3390/toxins14020149

Cited by 12 publications

(6 citation statements)

References 66 publications

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“…The interaction between CTX and the cell membrane is known to be closely related to the types of CTX, namely S-type and P-type. The insertion of S-type CTX into the cell membrane was primarily relying on the structural adaptation of loop II 62 . On the other hand, loops II and III of the P-type CTX were found to be more critical in lipid membrane penetration 63 .…”

Section: Resultsmentioning

confidence: 99%

Unveiling the functional epitopes of cobra venom cytotoxin by immunoinformatics and epitope-omic analyses

Hiu

Fung

Tan

et al. 2023

Sci Rep

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Approximate 70% of cobra venom is composed of cytotoxin (CTX), which is responsible for the dermonecrotic symptoms of cobra envenomation. However, CTX is generally low in immunogenicity, and the antivenom is ineffective in attenuating its in vivo toxicity. Furthermore, little is known about its epitope properties for empirical antivenom therapy. This study aimed to determine the epitope sequences of CTX using the immunoinformatic analyses and epitope-omics profiling. A conserved CTX was used in this study to determine its T-cell and B-cell epitope sequences using immunoinformatic tools and molecular docking simulation with different Human Leukocyte Antigens (HLAs). The potential T-cell and B-cell epitopes were 'KLVPLFY,' 'CPAGKNLCY,' 'MFMVSTPTK,' and 'DVCPKNSLL.' Molecular docking simulations disclosed that the HLA-B62 supertype exhibited the greatest binding affinity towards cobra venom cytotoxin. The namely L7, G18, K19, N20, M25, K33, V43, C44, K46, N47, and S48 of CTX exhibited prominent intermolecular interactions with HLA-B62. The multi-enzymatic-limited-digestion/liquid chromatography-mass spectrometry (MELD/LC–MS) also revealed three potential epitope sequences as 'LVPLFYK,' 'MFMVS,' and ‘TVPVKR’. From different epitope mapping approaches, we concluded four potential epitope sites of CTX as ‘KLVPLFYK’, ‘AGKNL’, ‘MFMVSTPKVPV’ and ‘DVCPKNSLL’. Site-directed mutagenesis of these epitopes confirmed their locations at the functional loops of CTX. These epitope sequences are crucial to CTX’s structural folding and cytotoxicity. The results concluded the epitopes that resided within the functional loops constituted potential targets to fabricate synthetic epitopes for CTX-targeted antivenom production.

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Section: Resultsmentioning

confidence: 99%

Unveiling the functional epitopes of cobra venom cytotoxin by immunoinformatics and epitope-omic analyses

Hiu

Fung

Tan

et al. 2023

Sci Rep

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“…These are cytotoxin 3 (Uniprot code P24780) with a Val48-Leu49 pair and cytotoxin 2 N. kaouthia (Uniprot code P01445) with Leu 48-Val 49. X-ray analysis of the crystal structure of the toxin, namely its 2Fo-Fc electron density map at 2σ level for 47–52 region, allowed us to conclude that the correct order of the residues in this fragment is Leu48-Val49 [ 37 ]. Thus, the toxin is identical to cytotoxin 2 from N. kaouthia venom.…”

Section: Resultsmentioning

confidence: 99%

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop

Dubovskii

Ignatova

Алексеева

et al. 2022

Toxins

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In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.

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“…The first structure dates back to 1990 [ 24 ] and it represents the refinement of a model obtained in 1987 [ 25 ]. The most recent structure was published in 2022 [ 26 ]. The noted discrepancies between the X-ray and NMR structures of CTs [ 2 ] have been resolved by applying higher-field NMR spectrometers.…”

Section: Ct Structure and Biological Effectsmentioning

confidence: 99%

“…The recent NMR structures take into consideration the long-lived tightly bound water molecule in the second loop [ 27 , 28 ], and are determined using heteronuclear 13 C, 15 N-chemical shifts obtained either at the natural content of these isotopes [ 28 ] or at recombinantly produced isotope-labeled CTs [ 29 , 30 ]. The X-ray diffraction structure of one CT ( Figure 1 a) (PDB code 7QHI) [ 26 ] is almost identical to the NMR structure of this toxin (6RC7) [ 31 ].…”

Section: Ct Structure and Biological Effectsmentioning

confidence: 99%

Specific Amino Acid Residues in the Three Loops of Snake Cytotoxins Determine Their Membrane Activity and Provide a Rationale for a New Classification of These Toxins

Dubovskii,

Utkin

2024

Toxins

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Cytotoxins (CTs) are three-finger membrane-active toxins present mainly in cobra venom. Our analysis of the available CT amino acid sequences, literature data on their membrane activity, and conformational equilibria in aqueous solution and detergent micelles allowed us to identify specific amino acid residues which interfere with CT incorporation into membranes. They include Pro9, Ser28, and Asn/Asp45 within the N-terminal, central, and C-terminal loops, respectively. There is a hierarchy in the effect of these residues on membrane activity: Pro9 > Ser28 > Asn/Asp45. Taking into account all the possible combinations of special residues, we propose to divide CTs into eight groups. Group 1 includes toxins containing all of the above residues. Their representatives demonstrated the lowest membrane activity. Group 8 combines CTs that lack these residues. For the toxins from this group, the greatest membrane activity was observed. We predict that when solely membrane activity determines the cytotoxic effects, the activity of CTs from a group with a higher number should exceed that of CTs from a group with a lower number. This classification is supported by the available data on the cytotoxicity and membranotropic properties of CTs. We hypothesize that the special amino acid residues within the loops of the CT molecule may indicate their involvement in the interaction with non-lipid targets.

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Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling

Cited by 12 publications

References 66 publications

Unveiling the functional epitopes of cobra venom cytotoxin by immunoinformatics and epitope-omic analyses

Unveiling the functional epitopes of cobra venom cytotoxin by immunoinformatics and epitope-omic analyses

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop

Specific Amino Acid Residues in the Three Loops of Snake Cytotoxins Determine Their Membrane Activity and Provide a Rationale for a New Classification of These Toxins

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