Rigidity of loop 1 contributes to equipotency of globular and ribbon isomers of α-conotoxin AusIA
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ABSTRACT:
SUBMITTER: Ho T
PROVIDER: S-EPMC8578332 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed ?-conotoxin (?-CTx) that demonstrates potent inhibition of rat ?3?2-nicotinic acetylcholine receptors (r?3?2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of r?3?2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by ?-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of ?-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2-C16 and C3-C8 with an estimated 12.5% ?-helical content. In contrast, ?-CTx MII, which has a native fold of C2-C8 and C3-C16, has an estimated 38.1% ?-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) ?-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of r?3?2-nAChR subtypes.
| S-EPMC6950571 | biostudies-literature
Project description:Lysophosphatidic acid receptor 6 (LPAR6) is a G-protein coupled receptor (GPCR) involved in hair development and cytoskeleton formation in mammals. Its proliferation is implicated in several forms of cancer including liver cancer, squamous cell carcinoma and metastatic prostate cancer. Current study emphasizes the isolation of competitive non-lipid and stable peptide antagonists for Lysophosphatidic acid ligand. A total of 148 conotoxin structures were characterized for their binding abilities against LPAR6. Subsequently, top 10 conotoxins were selected on the basis of binding energy values, residual contributions and conformational cluster saturations. BuIA (a member of Alpha- conotoxins family), contryphan-R and contryphan-Lo (Synthetic class) conotoxins, exhibiting efficient binding parameters were subjected to molecular dynamics simulation assays and topology analysis. We propose that BuIA might be a potent antagonist due to its predominant binding at the extracellular region of LPAR6. Current study provides a backbone for understanding structural and functional insights of LPAR6 and findings of this study may be helpful in designing novel therapeutic targets for the treatment of cancers caused by elevated LPAR6 expression.
| S-EPMC5718415 | biostudies-literature
Project description:Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to approximately 50%. The maximal inhibition was stoichiometry-dependent because altering alpha3:beta4 RNA injection ratios either increased AuIB(ribbon) efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity alpha3beta4 nAChRs with corresponding EC(50) values of 22.6 and 176.9 microM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both alpha3beta4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha3beta4 nAChRs competitively, whereas AuIB(globular) inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.
| S-EPMC2903359 | biostudies-literature
Project description:Electro-ribbon actuators are lightweight, flexible, high-performance actuators for next generation soft robotics. When electrically charged, electrostatic forces cause the electrode ribbons to progressively zip together through a process called dielectrophoretic liquid zipping (DLZ), delivering contractions of more than 99% of their length. Electro-ribbon actuators exhibit pull-in instability, and this phenomenon makes them challenging to control: below the pull-in voltage threshold, actuator contraction is small, while above this threshold, increasing electrostatic forces cause the actuator to completely contract, providing a narrow contraction range for feedforward control. We show that application of a time-varying voltage profile that starts above pull-in threshold, but subsequently reduces, allows access to intermediate steady-states not accessible using traditional feed-forward control. A modified proportional-integral closed-loop controller is proposed (Boost-PI), which incorporates a variable boost voltage to temporarily elevate actuation close to, but not exceeding, the pull-in voltage threshold. This primes the actuator for zipping and drastically reduces rise time compared with a traditional PI controller. A multi-objective parameter-space approach was implemented to choose appropriate controller gains by assessing the metrics of rise time, overshoot, steady-state error, and settle time. This proposed control method addresses a key limitation of the electro-ribbon actuators, allowing the actuator to perform staircase and oscillatory control tasks. This significantly increases the range of applications which can exploit this new DLZ actuation technology.
| S-EPMC7805931 | biostudies-literature
Project description:The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson's and Alzheimer's diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(-) or α4(+)α4(-) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)3(β2)2 nAChR was deduced using the information from the cryo-electron structure of (α4)3(β2)2 nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(-) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)3(β2)2 nAChR.
| S-EPMC8469569 | biostudies-literature
Project description:Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.
| S-EPMC6669574 | biostudies-literature
Project description:Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure-function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 α-conotoxin TxIA, a potent blocker at α3β2 and α7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli. Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1-2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3β2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure-function analysis for a ribbon 4/7-α-conotoxin at α7 and α3β2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.
| S-EPMC6554660 | biostudies-literature
Project description:Selected amyloid structures available in the Protein Data Bank have been subjected to a comparative analysis. Classification is based on the distribution of hydrophobicity in amyloids that differ with respect to sequence, chain length, the distribution of beta folds, protofibril structure, and the arrangement of protofibrils in each superfibril. The study set includes the following amyloids: Aβ (1-42), which is listed as Aβ (15-40) and carries the D23N mutation, and Aβ (11-42) and Aβ (1-40), both of which carry the E22Δ mutation, tau amyloid, and α-synuclein. Based on the fuzzy oil drop model (FOD), we determined that, despite their conformational diversity, all presented amyloids adopt a similar structural pattern that can be described as a ribbon-like micelle. The same model, when applied to globular proteins, results in structures referred to as "globular micelles," emerging as a result of interactions between the proteins' constituent residues and the aqueous solvent. Due to their composition, amyloids are unable to attain entropically favorable globular forms and instead attempt to limit contact between hydrophobic residues and water by producing elongated structures. Such structures typically contain quasi hydrophobic cores that stretch along the fibril's long axis. Similar properties are commonly found in ribbon-like micelles, with alternating bands of high and low hydrophobicity emerging as the fibrils increase in length. Thus, while globular proteins are generally consistent with a 3D Gaussian distribution of hydrophobicity, the distribution instead conforms to a 2D Gaussian distribution in amyloid fibrils.
| S-EPMC6930452 | biostudies-literature
Project description:Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.
| S-EPMC2829941 | biostudies-literature
Project description:Parkinson's disease is a debilitating movement disorder characterized by altered levels of alpha(6)beta(2) * ( * indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. alpha-Conotoxin MII (alpha-CTx MII) is a highly useful ligand to probe alpha(6)beta(2) nAChRs structure and function, but it does not discriminate among closely related alpha(6) * nAChR subtypes. Modification of the alpha-CTx MII primary sequence led to the identification of alpha-CTx MII[E11A], an analog with 500-5300-fold discrimination between alpha(6) * subtypes found in both human and non-human primates. alpha-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity alpha(6) nAChR, a subtype that is selectively lost in Parkinson's disease. Here, we present the three-dimensional solution structure for alpha-CTx MII[E11A] as determined by two-dimensional (1)H NMR spectroscopy to 0.13+/-0.09A backbone and 0.45+/-0.08A heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of alpha-CTx MII[E11A] relative to other members of the alpha-CTx family may be responsible for its exceptionally high affinity for alpha6alpha4beta2 * nAChR as well as discrimination between alpha(6)beta(2) and alpha(3)beta(2) containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for alpha(6) * nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson's disease.
| S-EPMC2754775 | biostudies-literature