Biochemistry
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Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus. In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). ⋯ For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 μM, respectively. When [(3)H]AziPm (7 μM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.
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Animal venoms contain a fascinating array of divergent peptide toxins that have cross-activities on different types of voltage-gated ion channels. However, the underlying mechanism remains poorly understood. Jingzhaotoxin-III (JZTX-III), a 36-residue peptide from the tarantula Chilobrachys jingzhao, is specific for Nav1.5 and Kv2.1 channels over the majority of other ion channel subtypes. ⋯ The bioactive surfaces of JZTX-III interacting with Kv2.1 and Nav1.5 are only partially overlapping. These results strongly supported the hypothesis that animal toxins might use partially overlapping bioactive surfaces to target the voltage-sensor paddles of two different types of ion channels. Increasing our understanding of the molecular mechanisms of toxins interacting with voltage-gated sodium and potassium channels may provide new molecular insights into the design of more potent ion channel inhibitors.
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Currently, several models for predicting the secondary structure of RNA exist, one of which is free energy minimization using the Nearest Neighbor Model. This model predicts the lowest-free energy secondary structure from a primary sequence by summing the free energy contributions of the Watson-Crick nearest neighbor base pair combinations and any noncanonical secondary structure motif. The Nearest Neighbor Model also assumes that the free energy of the secondary structure motif is dependent solely on the identities of the nucleotides within the motif and the motif's nearest neighbors. ⋯ In each of these oligonucleotides, the hairpin loop sequence and the adjacent base pairs were held constant, while the first or second non-nearest neighbors were varied. The experimental results show that the thermodynamic contributions of the hairpin loop were dependent upon the identity of the first non-nearest neighbor, while the second non-nearest neighbor had a less obvious effect. These results were then used to create an updated model for predicting the thermodynamic contributions of a hairpin loop to the overall stability of the stem-loop structure.
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The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [³H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. ⋯ To further define GABA(A)R etomidate binding sites, we now use [³H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [³H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [³H]TDBzl-etomidate and [³H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA(A)R.
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Comparative Study
The role of conserved Glu residue on cyclotide stability and activity: a structural and functional study of kalata B12, a naturally occurring Glu to Asp mutant.
Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). ⋯ Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.