Characterization of the Native Disulfide Isomers of the Novel χ-Conotoxin PnID: Implications for Further Increasing Conotoxin Diversity.

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers.

Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein.

As the only major retrograde transporter along microtubules, cytoplasmic dynein plays crucial roles in the intracellular transport of organelles and other cargoes. Central to the function of this motor protein complex is dynein intermediate chain (IC), which binds the three dimeric dynein light chains at multivalent sites, and dynactin p150Glued and nuclear distribution protein (NudE) at overlapping sites of its intrinsically disordered N-terminal domain. The disorder in IC has hindered cryo-electron microscopy and X-ray crystallography studies of its structure and interactions. Here we use a suite of biophysical methods to reveal how multivalent binding of the three light chains regulates IC interactions with p150Glued and NudE. Using IC from Chaetomium thermophilum, a tractable species to interrogate IC interactions, we identify a significant reduction in binding affinity of IC to p150Glued and a loss of binding to NudE for constructs containing the entire N-terminal domain as well as for full-length constructs when compared to the tight binding observed with short IC constructs. We attribute this difference to autoinhibition caused by long-range intramolecular interactions between the N-terminal single α-helix of IC, the common site for p150Glued, and NudE binding, and residues closer to the end of the N-terminal domain. Reconstitution of IC subcomplexes demonstrates that autoinhibition is differentially regulated by light chains binding, underscoring their importance both in assembly and organization of IC, and in selection between multiple binding partners at the same site.

Motor proteins are the freight trains of the cell, transporting large molecular cargo from one location to another using an array of 'roads' known as microtubules. These hollow tubes are oriented, with one extremity (the plus-end) growing faster than the other (the minus-end). While over 40 different motor proteins travel towards the plus-end of microtubules, just one is responsible for moving cargo in the opposite direction. This protein, called dynein, performs a wide range of functions which must be carefully regulated, often through changes in the shape and interactions of various dynein segments. The intermediate chain is one of the essential subunits that form dynein, and it acts as a binding site for a range of molecular actors. In particular, it connects the three other dynein subunits (known as the light chains) to the dynein heavy chain containing the motor domain. It also binds to two non-dynein proteins: NudE, which helps to organise microtubules, and the p150^Glued region of dynactin, a protein required for dynein activity. Despite their distinct roles, p150^Glued and NudE attach to the same region of the intermediate chain, a highly flexible 'unstructured' segment which is difficult to study. How the binding of p150^Glued and NudE is regulated has therefore remained unsolved. In response, Jara et al. decided to investigate how the three dynein light chains may help to control interactions between the intermediate chain and non-dynein proteins. They used more stable versions of dynein, NudE and dynactin (from a fungus that grows at high temperatures) to produce the various subcomplexes formed by the intermediate chain, the three dynein light chains, and parts of p150^Glued and NudE. A suite of biophysical techniques was applied to study these structures, as they are challenging to capture using traditional approaches. This revealed that the unstructured region of the intermediate chain can fold back on itself, bringing together its two extremities; such folding blocks the p150^Glued and NudE binding site. This obstruction is cleared when the light chains bind to the intermediate chain, demonstrating how these three subunits can regulate dynein activity. In humans, mutations in dynein are associated with a range of serious neurological and muscular diseases. The work by Jara et al. brings new insight into the way this protein works; more importantly, it describes how to combine several biophysical techniques to study non-structured proteins, offering a blueprint that is likely to be relevant for a wide range of scientists.

Interplay of Disorder and Sequence Specificity in the Formation of Stable Dynein-Dynactin Complexes.

Cytoplasmic dynein is a eukaryotic motor protein complex that, along with its regulatory protein dynactin, is essential to the transport of organelles within cells. The interaction of dynein with dynactin is regulated by binding between the intermediate chain (IC) subunit of dynein and the p150Glued subunit of dynactin. Even though in the rat versions of these proteins this interaction primarily involves the single α-helix region at the N-terminus of the IC, in Drosophila and yeast ICs the removal of a nascent helix (H2) downstream of the single α-helix considerably diminishes IC-p150Glued complex stability. We find that for ICs from various species, there is a correlation between disorder in H2 and its contribution to binding affinity, and that sequence variations in H2 that do not change the level of disorder show similar binding behavior. Analysis of the structure and interactions of the IC from Chaetomium thermophilum demonstrates that the H2 region of C. thermophilum IC has a low helical propensity and establishes that H2 binds directly to the coiled-coil 1B (CC1B) domain of p150Glued, thus explaining why H2 is necessary for tight binding. Isothermal titration calorimetry, circular dichroism, and NMR studies of smaller CC1B constructs localize the region of CC1B most essential for a tight interaction with IC. These results suggest that it is the level of disorder in H2 of IC along with its charge, rather than sequence specificity, that underlie its importance in initiating tight IC-p150Glued complex formation. We speculate that the nascent H2 helix may provide conformational flexibility to initiate binding, whereas those species that have a fully folded H2 have co-opted an alternative mechanism for promoting p150Glued binding.

Characterization of Three Venom Peptides from the Spitting Spider Scytodes thoracica.

We present the solution-state NMR structures and preliminary functional characterizations of three venom peptides identified from the spitting spider Scytodes thoracica. Despite little sequence identity to other venom peptides, structural characterization reveals that these peptides contain an inhibitor cystine knot motif common to many venom peptides. These are the first structures for any peptide or protein from spiders of the Scytodidae family. Many venom peptides target neuronal ion channels or receptors. However, we have not been able to determine the target of these Scytodes peptides so we can only state with certainty the channels and receptors that they do not target.

Solution structures of two homologous venom peptides from Sicarius dolichocephalus.

We present solution-state NMR structures for two putative venom peptides from Sicarius dolichocephalus. These peptides were identified from cDNA libraries created from venom gland mRNA and then recombinantly expressed. They are the first structures from any species of Sicarius spiders, and the first peptide structures for any haplogyne spiders. These peptides are homologous to one another, and while they have at most only 20% sequence identity with known venom peptides their structures follow the inhibitor cystine knot motif that has been found in a broad range of venom peptides.

Solution structure of the U2 snRNP protein Rds3p reveals a knotted zinc-finger motif.

Rds3p, a component of the U2 snRNP subcomplex SF3b, is essential for pre-mRNA splicing and is extremely well conserved in all eukaryotic species. We report here the solution structure of Rds3p, which reveals an unusual knotted fold unrelated to previously known knotted proteins. Rds3p has a triangular shape with a GATA-like zinc finger at each vertex. Pairs of cysteines contributing to each finger are arranged nonsequentially in a permuted arrangement reminiscent of domain-swapping but which here involves segments of subdomains within a single chain. We suggest that the structure arose through a process of segment swapping after gene duplication. The fingers are connected through beta-strands and loops, forming an overall topology strongly resembling a "triquetra knot." The conservation and surface properties of Rds3p suggest that it functions as a platform for protein assembly within the multiprotein SF3b complex of U2 snRNP. The recombinant protein used for structure determination is biologically active, as it restores splicing activity in a yeast splicing extract depleted of native Rds3p.

Quantification of phosphocholine and glycerophosphocholine with 31P edited 1H NMR spectroscopy.

Choline and the related compounds phosphocholine (PC) and glycerophosphocholine (GPC) are considered to be important metabolites in oncology. Past studies have demonstrated correlations linking the relative ratios and concentrations of these metabolites with the development and progression of cancer. Currently, in vivo and tissue ex vivo magnetic resonance spectroscopy methods have mostly centered on measuring the total concentration of these metabolites and have difficulty in differentiating between them. Here, a new scheme that uses (31)P edited (1)H spectroscopy to quantify the concentrations of choline, PC and GPC in biological samples is reported and its applicability is demonstrated using samples of human brain tumor extracts. This method is particularly well-suited for analytical situations where the PC and GPC resonances are not sufficiently resolved and/or are obscured by other metabolites. Consequently, this scheme has the potential to be used for the analysis of choline compounds in ex vivo tissue samples.

Single-scan longitudinal relaxation measurements in high-resolution NMR spectroscopy.

Several single-scan experiments for the measurement of the longitudinal relaxation time (T1) are proposed. These experiments result in fast and accurate determinations of the relaxation rate, are relatively robust to pulse imperfections, and preserve information about the chemical shift. The method used in these experiments is to first encode the T1 values as a spatial variation of the magnetization and then to read out this variation either by applying a weak gradient during acquisition or by sequentially observing different slices of the sample. As a result, it is possible to reduce the time necessary to determine the T1 values by one or two orders of magnitude. This time saving comes at the expense of the signal-to-noise level of the resulting spectrum and some chemical shift resolution.