¹H Nuclear‐Magnetic‐Resonance Studies of the Conformation of Cardiotoxin V^II2 from Naja mossambica mossambica

Abstract

The membrane toxin V"2 from the venom of Naja mossamhica niossambica was investigated in aqueous solution by one-dimensional and two-dimensional high-resolution nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization included identification of the complete spin systems for several amino acid residues, nuclear Overhauser effect measurements, the use of chemically induced dynamic nuclear polarization and studies of the pH dependence of the N M R spectrum. Data from homologous toxins, in particular direct lytic factor 12B from Haemachatus haenzachatus, were used to establish assignments of aromatic and methyl proton resonances. From these experiments a short, triple-stranded fragment of antiparallel p structure could be determined, which includes the residues 23 -27, 43 -46 and 60 -62. Furthermore, the nuclear Overhauser effect measurements indicate close proximity in the protein conformation of the aromatic rings of Trp-14, Tyr-25 and Tyr-59, and the side chain of Ile-46.Keen interest in the toxins from the venoms of snakes belonging to the family Elapidae (cobra, coral snake, sea snake, etc.) has generated an extensive literature [l -31. Three classes of toxins are usually distinguished: long neurotoxins, short neurotoxins, and membrane toxins. These three classes form a group of homologous proteins with 71 -74, 60 -62, and 60-62 amino acid residues, respectively, arranged in a single polypeptide chain. If one excludes an extra disulfide bridge in the long neurotoxins, all of the approximately 80 toxins sequenced so far have four disulfide bridges linking half-cysteine residues which have homologous locations in the sequence. In addition seven other amino acid residues are located at homologous positions. Within a class or subclass of toxins, homology is even more extensive and can be as high as 60-70 % (e.g. membrane toxins from the genus Nuja). In spite of the extensive homology, there are pronounced differences in the mode of action of the different types of toxins. Neurotoxins bind to a protein receptor at the post-synaptic level and block acetylcholine reception [4]. The action of membrane'toxins results in a variety of effects including hemolysis, cytotoxieity, depolarization of excitable membrane and modulation of membranal enzyme activity [2,5]. This varied phenomenology has produced a profusion of trivial names such as cardiotoxin, cytotoxin, and direct lytic factor. A common trait of the actions of the different membrane toxins at the cellular level appears to be binding to the cell membrane with disturbance of its organization and function [5 -71.A variety of approaches have been used to investigate the spatial structures of snake neurotoxins and cardiotoxins [I, 21. These have included structure-activity modelling studies employing in part Chou-Fasman-type predictions [8 -lo], Abbreviations. CD, circular dichroism; 6, chemical shift; lytic factor 12B, direct lytic factor 12B from Haemachatus haemachaius; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; ppi...

Sequential Individual Resonance Assignments in the 1H Nuclear‐Magnetic‐Resonance Spectrum of Cardiotoxin VII 2 from Naja mossambica mossambica

“…3 shows a 500-MHz NOESY spectrum of toxin V"2. It can be Amino acids or individual resonances which were previously assigned [8]. Small differences between the chemical shifts in the present and the previous publication [8] must be attributed t o the improved resolution at 500 MHz.…”

“…' The three Met L l I , singlets were alrcady identified in earlicr experiments [8] but could not be individually assigned. The linc at 1.97 ppm was tentatively assigned to seen that this spectrum contains a large number of well defined and quite intense cross peaks, which indicates that the protein adopts a well defined conformation with restricted mobility for the bulk of the polypeptide backbone and many of the amino acid side chains.…”

“…1 1 below), indicating that the resonances of all, or nearly all, the amino acid residues can be resolved in the 2 D N M R spectra of this protein. In the following, individual assignments of the resonances in these spectra are described and documented for all those residues for which this data was not previously obtained [8].…”

“…In earlier experiments [8] 360-MHz 'H N M R spectra of toxin V"2 in 2 H 2 0 solution were analyzed and a limited number of resonance lines could be assigned (see Table 1). The previous observation that these spectra contain numerous well defined peaks is conrirmed by the 500-MHz 'H SECSY spectra of Fig.…”

“…Physical-chemical studies of snake toxin conformations (see [8] for a more comprehensive survey) include numerous NMR investigations of static and dynamic aspects of the threedimensional protein structures [9 -241. Most of these NMR studies concentrated on neurotoxins and, to some degree, the interpretation of the NMR data relied on the known crystal structure of erabutoxin b [25-281. In the present paper we make use of new procedures recently introduced [29 -341 for obtaining nearly complete resonance assignments in the 'H NMR spectrum of toxin V"2.…”