Hervé Darbon

Evolutionary origin of inhibitor cystine knot peptides

The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel have been found to adopt an ICK fold. We have isolated and identified both cDNA and genomic clones for this family of ICK peptides from the scorpion Opistophthalmus carinatus. The gene characterized by three well‐delineated exons respectively coding for three structural and functional domains in the toxin precursors illustrates the correlation between exon and module as suggested by the “exon theory of genes.” Based on the analysis of precursor organization and gene structure combined with the 3‐D fold and functional data, our results highlight a common evolutionary origin for ICK peptides from animals. In contrast, ICK peptides from plant and fungus might be independently evolved from another ancestor.

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded.

Measles virus is a negative‐sense, single‐stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N‐terminal domain, and of an intrinsically disordered C‐terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C‐terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in NTAIL, an α‐helical molecular recognition element (α‐MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small‐angle X‐ray scattering, we have derived a low‐resolution structural model of the complex between XD and NTAIL, which shows that most of NTAIL remains disordered in the complex despite P‐induced folding within the α‐MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N‐terminal region of NTAIL, and of a bulky globular region, corresponding to XD and to the C terminus of NTAIL (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that NTAIL has an additional site (aa 517–525) involved in binding to XD but not in the unstructured‐to‐structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.

K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges that was initially isolated from the venom of the Asian scorpion Orthochirus scrobiculosus. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+-activated K+ channels in vitro. In the present paper, we report that OSK1 is lethal in mice by intracerebroventricular injection, with a LD50 (50% lethal dose) value of 2 microg/kg. OSK1 blocks K(v)1.1, K(v)1.2, K(v)1.3 channels potently and K(Ca)3.1 channel moderately, with IC50 values of 0.6, 5.4, 0.014 and 225 nM respectively. Structural analogues of OSK1, in which we mutated positions 16 (Glu16-->Lys) and/or 20 (Lys20-->Asp) to amino acid residues that are conserved in all other members of the alpha-KTx3 toxin family except OSK1, were also produced and tested. Among the OSK1 analogues, [K16,D20]-OSK1 (OSK1 with Glu16-->Lys and Lys20-->Asp mutations) shows an increased potency on K(v)1.3 channel, with an IC50 value of 0.003 nM, without loss of activity on K(Ca)3.1 channel. These data suggest that OSK1 or [K16,D20]-OSK1 could serve as leads for the design and production of new immunosuppressive drugs.

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a β-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton‐gated cation channels

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 1H 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 β‐sheet and the β‐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.

A new fold in the scorpion toxin family, associated with an activity on a ryanodine-sensitive calcium channel.

We determined the structure in solution by 1H two‐dimensional NMR of Maurocalcine from the venom of Scorpio maurus. This toxin has been demonstrated to be a potent effector of ryanodyne‐sensitive calcium channel from skeletal muscles. This is the first description of a scorpion toxin which folds following the Inhibitor Cystine Knot fold (ICK) already described for numerous toxic and inhibitory peptides, as well as for various protease inhibitors. Its three dimensional structure consists of a compact disulfide‐bonded core from which emerge loops and the N‐terminus. A double‐stranded antiparallel β‐sheet comprises residues 20–23 and 30–33. A third extended strand (residues 9–11) is perpendicular to the β‐sheet. Maurocalcine structure mimics the activating segment of the dihydropyridine receptor II‐III loop and is therefore potentially useful for dihydropyridine receptor/ryanodine receptor interaction studies. Proteins 2000;40:436–442.

SOLUTION STRUCTURE OF MAUROTOXIN, A SCORPION TOXIN FROM SCORPIO MAURUS, WITH HIGH AFFINITY FOR VOLTAGE-GATED POTASSIUM CHANNELS

Maurotoxin (MTX), purified from the scorpionid Scorpio maurus is a potent ligand for potassium channels. It shows a broad specificity as being active on Kv1.1 (Kd = 37 nM), Kv1.2 (Kd = 0.8 nM), Kv1.3 (Kd = 150 nM) voltage‐gated potassium channels, as well as on small‐conductance calcium‐activated potassium channels. It has a unique disulfide pairing among the scorpion toxins family. The solution structure of MTX has been determined by 2D‐NMR techniques, which led to the full description of its 3D conformation: a bended helix from residues 6 to 16 connected by a loop to a two‐stranded antiparallel β sheet (residues 23 to 26 and 28 to 31). The interaction of MTX with the pore region of the Kv1.2 potassium channel has been modeled according to their charge anisotropy. The structure of MTX is similar to other short scorpion toxins despite its peculiar disulfide pairing. Its interaction with the Kv1.2 channel involves a dipole moment, which guides and orients the toxin onto the pore, toward the binding site, and which thus is responsible for the specificity. Proteins 29:321–333, 1997.

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein

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, (NTAIL, aa 401–525), is also presented. XD is an all α 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 α‐helical XD‐binding region of NTAIL. Secondary chemical shift values for full‐length NTAIL 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 NTAIL. Titration experiments with unlabeled XD showed that the transient α‐helical conformation of NTAIL is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483–506 of NTAIL are in intermediate exchange with XD, while the 475–482 and 507–525 regions are in fast exchange. The NTAIL resonance behavior in the titration experiments is consistent with a complex binding model with more than two states. Copyright

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH ‐R2 fragment and its antigen, the dye Reactive Red‐6 (RR6) has been explored. The data show clearly that most of the VHH‐R2 population at 80°C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH‐R2 and the dye remained the same up to 80°C. Interestingly, addition of the dye to the denatured VHH‐R2 at 80°C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH‐R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established “induced fit” mechanism of antibody–antigen interactions at ambient temperature. However, the main difference with the “induced fit” mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications. Proteins 2005.

An unusual fold for potassium channel blockers: NMR structure of three toxins from the scorpion Opisthacanthus madagascariensis

The Om-toxins are short peptides (23-27 amino acids) purified from the venom of the scorpion Opisthacanthus madagascariensis. Their pharmacological targets are thought to be potassium channels. Like Csalpha/beta (cystine-stabilized alpha/beta) toxins, the Om-toxins alter the electrophysiological properties of these channels; however, they do not share any sequence similarity with other scorpion toxins. We herein demonstrate by electrophysiological experiments that Om-toxins decrease the amplitude of the K+ current of the rat channels Kv1.1 and Kv1.2, as well as human Kv1.3. We also determine the solution structure of three of the toxins by use of two-dimensional proton NMR techniques followed by distance geometry and molecular dynamics. The structures of these three peptides display an uncommon fold for ion-channel blockers, Csalpha/alpha (cystine-stabilized alpha-helix-loop-helix), i.e. two alpha-helices connected by a loop and stabilized by two disulphide bridges. We compare the structures obtained and the dipole moments resulting from the electrostatic anisotropy of these peptides with those of the only other toxin known to share the same fold, namely kappa-hefutoxin1.