Sequential assignment and structure determination of spider toxin .omega.-Aga-IVB

Abstract

The solution structure of a peptide toxin isolated from funnel web spider venom, omega-Aga-IVB, was determined by 2D NMR methods. omega-Aga-IVB is a high-affinity specific blocker of P-type voltage-dependent calcium channels. Nearly all of the proton resonances of this 48-residue protein were assigned using conventional 2D homonuclear NMR experiments. The three-dimensional structure of the molecule was determined by simulated annealing. The distance and dihedral restraints used in the structure calculations were derived from NOESY and COSY-type experiments, respectively. Mass spectrometric analysis of omega-Aga-IVB suggests that the protein contains four disulfide bonds. In the absence of chemical data to identify the pattern of cysteine pairing, the disulfide bonds of the toxin are proposed from the NMR data and subsequent structural calculations. The structure of the toxin can be described as a three-stranded anti-parallel beta sheet connected by flexible loops. A striking feature of the structure is that the C-terminal 10 residues of this protein adopt random coil conformations. Several positively charged amino acid side chains are found localized on one face of the molecule, in close proximity to the C-terminal tail. This observation has led us to propose a speculative model of the toxins blockade mechanism.

A common structural motif incorporating a cystine knot and a triple‐stranded β‐sheet in toxic and inhibitory polypeptides

“…With the exception of w-Aga IVB, which has a fourth disulfide bridge, the pattern of disulfide pairings is the same for each of these molecules. w-Aga IVB, the structure of which was determined recently (Yu et al, 1993), is included because its fourth disulfide bridge can be considered independent of the other 3 bridges, which form a cystine knot with the same topology as that of the other molecules in Figure 1 (although this s e q u e n c e motif s t r u c t u r a l m o t i f w C g T x GVIA Kalata BI CMTI-I EETI-I1 CP I w A g a IVB was not identified by Yu et al, 1993). EETI-I1 and CPI are included because broad topological and cystine knot similarities between these polypeptides and w-CgTx and CMTI-I have been noted previously (Bode et al, 1989;Le-Nguyen et al, 1990;Chiche et al, 1993;Pallaghy et al, 1993).…”

Structure and Sodium Channel Activity of an Excitatory I1-Superfamily Conotoxin,

“…In this conformation the switch from D to L configuration at Phe44 may be expected to have a significant effect on affinity and/or activity. The more structured region of ι-RXIA, the ICK motif, probably plays a common role in both ι-RXIA and ι-RXIA[L-Phe44] of mediating peptide binding to the appropriate site on the target in such a way as to allow the C-terminal tail to interact with neighboring residues.The functional importance of epimerization of a single amino acid near the C-terminus was also observed for ω-agatoxin IVB, which has a similar combination of an ICK motif core and an unstructured tail (13,38,40,41). In this 48-residue peptide, D-Ser46 was crucial for the inhibition of P-type calcium channels, whereas the L-Ser enantiomer was about 90-fold less active (40).…”

Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

“…4c). A similar model has been proposed for the mode of action of -Aga-IVB (35). Limited motion of the tail region within the membrane might minimize the loss of conformational entropy suffered by the toxin upon channel binding.…”