Proton NMR studies of the structural and dynamical effect of chemical modification of a single aromatic side-chain in a snake cardiotoxin. Relation to the structure of the putative binding site and the cytolytic activity of the toxin.
This paper presents the comparative comprehensive analysis of NMR structural parameters (NOEs, scalar coupling, chemical shifts) of toxin gamma, a cardiotoxin isolated from the venom of Naja nigricollis, and three chemical derivatives, i.e. the 2-nitrophenylsulphonyl (NPS)-Trp11, 3-nitro-Tyr22 and 3-nitro-Tyr51 derivatives. In previous work, the chemical modifications of single side chains have suggested that these aromatic residues, in association with several lysine residues, contributed to the cytotoxicity of toxin gamma. Analysis of these results based on the refined solution structure of the toxin has resulted in the proposal of a conserved phospholipid binding site through which cardiotoxins are likely to interact with the membrane of target cells. The present work shows that modifications of either the tryptophan residue or the tyrosine residues, which are within or near the proposed binding site, have no influence on the three-dimensional structure of the protein. On the other hand, the proton exchange study of the backbone amides indicates that the structural core of the protein is destabilized in the three derivatives. This corresponds to a decrease of the overall stability of the protein as indicated by the comparative solvent denaturation study of the unmodified toxin gamma and the Trp11 derivative. More specifically, the dynamics of the three-stranded beta sheet, a part of the structural core, are highly perturbed by the chemical modifications. This sheet was previously proposed as a part of the phospholipid binding site of cardiotoxins. The dynamical perturbation of this site appears to be correlated with the decrease in toxicity of the chemical derivatives.
Roumestand
C
C
Département d'Ingénierie et d'Etudes des Protéines (D.I.E.P.), C.E. Saclay, Gif-sur-Yvette, France.
Gilquin
B
B
Trémeau
O
O
Gatineau
E
E
Mouawad
L
L
Ménez
A
A
Toma
F
F
D000818:Animals; D001667:Binding, Competitive; D004179:Cobra Cardiotoxin Proteins / Q000737:chemistry*; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009578:Nitrobenzenes / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D012997:Solvents; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D004179:Cobra Cardiotoxin Proteins; D004546:Elapid Venoms; D009578:Nitrobenzenes; D011522:Protons; D012997:Solvents; D014443:Tyrosine; D014357:Trypsin