Paul K. Pallaghy

The cystine knot structure of ion channel toxins and related polypeptides

An increasing number of ion channel toxins and related polypeptides have been found to adopt a common structural motif designated the inhibitor cystine knot motif (Pallaghy P. K., Nielsen, K. J., Craik, D. J., Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded beta-sheet in toxic and inhibitory polypeptides. Protein Science 3, 1833-1839). These globular, disulfide-stabilized molecules come from phylogenetically diverse sources, including spiders, cone shells, plants and fungi, and have various functions, although many target voltage-gated ion-channels. The common motif consists of a cystine knot and a triple-stranded, anti-parallel beta-sheet. Examples of ion-channel toxins known to adopt this structure are the omega-conotoxins and omega-agatoxins, and, more recently, robustoxin, versutoxin and protein 5 from spiders, as well as kappa-conotoxin PVIIA and conotoxin GS from cone shells. The variations on the motif structure exemplified by these structures are described here. We also consider the sequences of several polypeptides that might adopt this fold, including SNX-325 from a spider, delta-conotoxin PVIA and the muO-conotoxins from cone shells, and various plant and fungal polypeptides. The interesting case of the two- and three-disulfide bridged binding domains of the cellobiohydrolases from the fungus Trichoderma reesei is also discussed. The compact and robust nature of this motif makes it an excellent scaffold for the design and engineering of novel polypeptides with enhanced activity against existing targets, or with activity against novel targets.

STRUCTURE-FUNCTION RELATIONSHIPS OF OMEGA -CONOTOXIN GVIA : SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES

The structure-function relationships of the N-type calcium channel blocker, ω-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus.

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel‐web spider Atrax robustus, has been determined from 2D 1H NMR data. Robustoxin is a polypeptide of 42 residues cross‐linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1–15, 8–20, 14–31 and 16–42 (a 1–4/2–6/3–7/5–8 pattern). The structure consists of a small three‐stranded, anti‐parallel β‐sheet and a series of interlocking γ‐turns at the C‐terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the ω‐conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non‐polar residues. Both of these structural features may play a role in its binding to the voltage‐gated sodium channel.

Solution structure of the cardiostimulant polypeptide anthopleurin-B and comparison with anthopleurin-A

BACKGROUND The polypeptide anthopleurin-B (AP-B) is one of a number of related toxins produced by sea anemones. AP-B delays inactivation of the voltage-gated sodium channel of excitable tissue. In the mammalian heart, this effect is manifest as an increase in the force of contraction. As a result, there is interest in exploiting the anthopleurins as lead compounds in the design of novel cardiac stimulants. Essential to this endeavour is a high-resolution solution structure of the molecule describing the positions of functionally important side chains. RESULTS AP-B exists in multiple conformations in solution as a result of cis-trans isomerization about the Gly40-Pro41 peptide bond. The solution structure of the major conformer of AP-B has been determined by two-dimensional 1H NMR at pH 4.5 and 25 degrees C. The core structure is a four-stranded, antiparallel beta-sheet (residues 2-4, 20-23, 34-37 and 45-48) and includes several beta-turns (6-9, 25-28, 30-33). Three loops connect the beta-strands, the longest and least well defined being the first loop, extending from residues 8-17. These features are shared by other members of this family of sea anemone toxins. The locations of a number of side chains which are important for the cardiac stimulatory activity of AP-B are well defined in the structures. CONCLUSIONS We have described the solution structure of AP-B and compared it with that of AP-A, from which it differs by substitutions at seven amino acid positions. It shares an essentially identical fold with AP-A yet is about 10-fold more active. Comparison of the structures, particularly in the region of residues essential for activity, gives a clearer indication of the location and extent of the cardioactive pharmacophore in these polypeptides.

Prior immunity helps to explain wave-like behaviour of pandemic influenza in 1918-9.

BackgroundThe ecology of influenza may be more complex than is usually assumed. For example, despite multiple waves in the influenza pandemic of 1918-19, many people in urban locations were apparently unaffected. Were they unexposed, or protected by pre-existing cross-immunity in the first wave, by acquired immunity in later waves, or were their infections asymptomatic?MethodsWe modelled all these possibilities to estimate parameters to best explain patterns of repeat attacks in 24,706 individuals potentially exposed to summer, autumn and winter waves in 12 English populations during the 1918-9 pandemic.ResultsBefore the summer wave, we estimated that only 52% of persons (95% credibility estimates 41-66%) were susceptible, with the remainder protected by prior immunity. Most people were exposed, as virus transmissibility was high with R0 credibility estimates of 3.10-6.74. Because of prior immunity, estimates of effective R at the start of the summer wave were lower at 1.57-3.96. Only 25-66% of exposed and susceptible persons reported symptoms. After each wave, 33-65% of protected persons became susceptible again before the next wave through waning immunity or antigenic drift. Estimated rates of prior immunity were less in younger populations (19-59%) than in adult populations (38-66%), and tended to lapse more frequently in the young (49-92%) than in adults (34-76%).ConclusionsOur model for pandemic influenza in 1918-9 suggests that pre-existing immune protection, presumably induced by prior exposure to seasonal influenza, may have limited the pandemic attack-rate in urban populations, while the waning of that protection likely contributed to recurrence of pandemic waves in exposed cities. In contrast, in isolated populations, pandemic attack rates in 1918-9 were much higher than in cities, presumably because prior immunity was less in populations with infrequent prior exposure to seasonal influenza. Although these conclusions cannot be verified by direct measurements of historical immune mechanisms, our modelling inferences from 1918-9 suggest that the spread of the influenza A (H1N1) 2009 pandemic has also been limited by immunity from prior exposure to seasonal influenza. Components of that immunity, which are measurable, may be short-lived, and not necessarily correlated with levels of HI antibody.

ROLE OF DISULFIDE BRIDGES IN THE FOLDING, STRUCTURE AND BIOLOGICAL ACTIVITY OF OMEGA -CONOTOXIN GVIA

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.

The cyclic contryphan motif CPxXPXC, a robust scaffold potentially useful as an ω-conotoxin mimic

: Contryphan-R, from venom of the cone-shell Conus radiatus, represents a novel cyclic peptide scaffold onto which residues may be grafted to mimic unrelated protein surfaces. Three substitutions were made at the x and X positions of the disulfide-bridged motif CPxXPXC, where X and x represent any L- and D-handed residues, respectively, P represents proline or hydroxyproline, and C a half-cystine. These substitutions were designed to mimic part of the pharmacophore of the unrelated globular polypeptide omega-conotoxin GVIA, which blocks N-type calcium channels. The structure of this engineered contryphan, YNK-contryphan-R ([D-Tyr4, Asn5, Lys7]contryphan-R), is shown to be similar to that of native contryphan-R (Pallaghy et al., Biochemistry, 1999, Vol. 38, pp. 13553-13559), confirming that the scaffold is robust with respect to the multiple substitutions. In particular, the alpha-beta bond vectors characterising the orientation of the side chains relative to the backbone are similar in contryphan-R, YNK-contryphan-R, and omega-conotoxin GVIA, which is the required result for a scaffold-based approach to molecular design. The solution structure of YNK-contryphan-R has an N-terminal, nonhydrogen-bonded, chain reversal centered on Hyp3-D-Trp4, and a C-terminal type I beta-turn. A minor form due to cis-trans isomerism of the Hyp2-Cys3 peptide bond is present in YNK-contryphan-R in a larger proportion than in contryphan-R. It is evident, particularly from the (3)J(HalphaHN) coupling constants, that YNK-contryphan-R is more flexible than contryphan-R, probably due to the absence in YNK-contryphan-R of the Pro-Trp packing present in the native molecule. Nevertheless, the structure confirms that cyclic peptide molecular designs can achieve the intended conformations.

Polypeptide ω-conotoxin GVIA as a basis for new analgesic and neuroprotective agents

The ω‐conotoxins are small polypeptides (of around 25 residues) cross‐linked by three disulfide bonds. At least two of these, ω‐conotoxins GVIA and MVIIA, are potent and selective blockers of N‐type voltage‐gated calcium channels. Administered intravenously, GVIA causes postural hypotension and blocks cardiac sympathetic and vagal reflexes, homeostatic cardiovascular effects that should normally be preserved. Administered intrathecally, MVIIA and GVIA are analgesic in acute, chronic, and neuropathic pain models, and protective following ischaemia‐induced neuronal injury. We have determined the three‐dimensional structure of GVIA and mapped onto that structure its calcium channel binding surface. This information is now being used in the structure‐based design of truncated and stabilised peptidic analogues of GVIA and in the development of peptidomimetic analogues. This article summarizes these data and outlines the strategies being pursued in the development of low molecular weight analogues for therapeutic applications. Drug Dev. Res. 46:206–218, 1999.

Helical structure and self-association in a 13 residue neuropeptide Y Y2 receptor agonist: relationship to biological activity

The solution structure and self-association behaviour of a 13 residue peptide analogue of the C-terminal region of human neuropeptide Y (NPY) have been investigated. NMR analysis of Ac[Leu(28,31)]NPY(24-36), a potent Y2 receptor agonist, shows that it is unstructured in aqueous solution at 5-20 degrees C, but forms a well-defined helix (encompassing residues 25-35) in 40% trifluoroethanol/water at 20 degrees C. Sedimentation experiments show that, in contrast to many peptides in aqueous trifluoroethanol, Ac[Leu(28,31)]NPY(24-36) associates to form a trimer or, more likely, a tetramer in 40% trifluoroethanol, even though it is monomeric in water. This is consistent with the observation of inter-molecular nuclear Overhauser enhancements in trifluoroethanol. Possible models of the associated form that are consistent with the NMR data are described. The relevance of the helical structure observed in trifluoroethanol to the structure of this peptide bound to the NPY Y2 receptor is discussed.

Understanding mortality in the 1918–1919 influenza pandemic in England and Wales

Please cite this paper as: Pearce et al. (2011) Understanding mortality in the 1918–1919 influenza pandemic in England and Wales. Influenza and Other Respiratory Viruses 5(2), 89–98.