In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

Acid-sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton-sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a spider toxin. The recombinant toxin was identical in all respects to the native peptide, and its three-dimensional structure in solution was determined by means of 1 H 2D NMR spectroscopy. Surface characteristics of PcTx1 provide insights on key structural elements involved in the binding of PcTx1 to ASIC1a channels. They appear to be localized in the ␤-sheet and the ␤-turn linking the strands, as indicated by electrostatic anisotropy calculations, surface charge distribution, and the presence of residues known to be implicated in channel recognition by other inhibitor cystine knot (ICK) toxins.Keywords: Spider toxin; NMR structure; ASIC; ICK motif Venoms of snakes, frogs, scorpions, spiders, cone snails, sea anemones, and some insects contain highly complex mixtures of bioactive peptides, many of which act primarily against ion channels. Peptide toxins isolated from these venoms have been invaluable tools for structural and physiological investigations of voltage-dependent Na + , Ca 2+ , and K + ion channels as well as for mechanosensitive channels (Olivera et al. 1991;Escoubas et al. 2000b;Lewis 2000). Numerous three-dimensional structures of peptide toxins acting on ion channels have been solved (Craik et al. 2001). In invertebrates, these peptides comprise ∼15 to 70 amino acids and are usually reticulated by disulfide bridges. A large number of the peptides studied to date belong to two major structural motifs. The first one is the cystine stabilized ␣/␤ scaffold (CS␣␤) comprising a short ␣-helix and a double-or triple-stranded antiparallel ␤-sheet stabilized by three or four disulfide bridges. This structural organization is essentially found in scorpion venom toxins acting on voltage-sensitive K + and Na + channels (Possani et al. 2000). The second type is the inhibitor cystine knot (ICK) motif which comprises several loops that emerge from a double-or triple-stranded antiparallel ␤-sheet structure reticulated by at least three disulfide bridges (Pallaghy et al. 1994;Norton and Pallaghy 1998). Two of the disulfide bridges together with the amino-acid backbone form a ring, Reprint requests to: Hervé Darbon, UMR 6098, CNRS, 31 ch. Joseph Aiguier, F-13402, Marseille Cedex 20, France; e-mail: herve@afmb.cnrsmrs.fr; fax: 33 (0)4-91-16-45-36; or Michel Lazdunski, IPMC-CNRS, 660 Route des Lucioles, F-06560, Valbonne, France; e-mail: ipmc@ipmc.cnrs.fr; fax: 33 (0)4-93-95-77-04. 4 These authors made equal contributions to this work. Abbreviations: PcTx1, psalmotoxin 1; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; COSY, correlation spe...