The omega-loop of cobra cytotoxins tolerates multiple amino acid substitutions

Self Cite

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.

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

Self Cite

“…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

Self Cite

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.

“…To check reproducibility of the simulations, three independent runs were performed for each toxin/lipid system listed in Table 3. In these starts, the orientations of the toxin molecules with respect the membrane surface were identical, but the distance from the center of mass of the toxin molecule to the center of the lipid bilayer was varied in the range 37-45 Å. a see Table 1 for the amino acid sequences of the toxins; b solution conformations of the major form of CT1No [22,39] or CT2Nk [37]; and c conformation of CT1No in detergent micelles [22].…”

Section: Molecular Dynamicsmentioning

confidence: 99%

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

Dubovskii

Dubova

Bourenkov

et al. 2022

Self Cite

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both “water” and “membrane” conformations of the central loop (loop-2) were determined by X-ray crystallography. The “water” conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The “membrane” conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step—the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their “water” NMR models. We found that the both toxins transform their “water” conformation of loop-2 into the “membrane” one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.