Search

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a &beta, hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known &beta, hairpin AMP family&mdash, tachyplesins and polyphemusins from the horseshoe crabs. The &beta, hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted &beta, hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known &beta, hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.

Lypd6 and Lypd6b are human three-finger proteins expressed in various tissues and sharing a high degree of sequence homology (~54%). Unlike other members of the Ly6/uPAR family, both proteins additionally bear extended N- and С-terminal sequences flanking the LU-domain. The role of these sequences is unclear. It is known that Lypd6 can increase the amplitude of nicotine-induced calcium currents in mouse trigeminal ganglion neurons. The Danio rerio fish Lypd6 is involved in the regulation of the Wnt/β-cathenin signaling pathway and the knockdown of the lypd6 expression leads to the impairment of embryonic development. The Lypd6b expression in X. laevis oocytes increased the sensitivity of nicotinic acetylcholine receptors to acetylcholine and increased their desensitization rate. Molecular mechanisms of Lypd6 and Lypd6b action as well as their spatial structures remain unknown. In this work, the genes encoding water-soluble variants of human Lypd6 and Lypd6b lacking N-terminal sequences (rLypd6 and rLypd6b) and Lypd6 bearing the N-terminal sequence (N-rLypd6) were obtained and expressed. The proteins were expressed in cytoplasmic inclusion bodies followed by solubilization under denaturing conditions and renaturation. The renaturation conditions were screened to optimize recombinant protein yields. The analysis of NMR spectra of recombinant N-rLypd6, rLypd6, and rLypd6b demonstrated that N-rLypd6 may not have a structured form. The production of milligram quantities of isotope-labeled rLypd6 and rLypd6b analogues allowed characterization of the secondary structure of these proteins and study of their intramolecular dynamics. It was found that rLypd6 and rLypd6b have structural elements typical for the Ly6/uPAR family, although with some unique features, particularly, an additional disulfide bond in the third loop and helical regions in the first and third loops.

Background: Nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel, which is widely distributed both in the central and peripheral nervous system, and in some of the non-neuronal tissues, including epithelium and immune cells. The pathophysiology of a number of diseases is associated with dysfunctions of this receptor, including neurodegenerative and mental disorders like Alzheimer disease (AD) and schizophrenia. The nicotinic receptor of α7 type (α7-nAChR) plays important role in the memory and learning processes and is inhibited by soluble aggregates of β-amyloid peptide (Aβ). Aβ1-42 is the most toxic form of the amyloid peptide. Methods: The water-soluble analogue of the ligand-binding extracellular domain of α7-nAChR was produced in Pichia pastoris and purified from a culture medium by Ni-NTA and size-exclusion chromatography. The equilibrium dissociation constant (Kd) of the complex of the recombinant domain with α-bungarotoxin was measured on the optical SPR biosensor Biacore 3000. Structures of the α7 domain alone and in the complex with oligomeric Aβ1-42 peptide were studied by cryoelectron microscopy (cryo-EM). Results: The α7 ligand-binding domain has an increased stability in solution, and demonstrates ligand-binding characteristics similar to those of the native receptor (Kd of the domain/α-bungarotoxin complex 28±2nM). Statistical analysis of the cryo-EM images of the individual domain particles revealed the presence of a pentameric structure, confirming intact subunit assembly. Unfortunately, the domain demonstrated the preferable orientation on grids with the top view. Nevertheless, the 3D structure of the domain with a height ~ 7 nm, external diameter of ~ 9 nm, and the pore diameter of ~ 2 nm was reconstructed at 8.5 Å resolution. 2D classification of the cryo-EM images of the domain particles in the complex with Aβ1-42 revealed the conformational changes appeared due to interaction with the amyloid peptide. Conclusion: Obtained results open new perspectives for structural studies of the nAChR ligand-binding domains in complex with the ligands which escape crystallization.

Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized &beta, structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake &alpha, neurotoxins WTX and NTII. Two different topologies of the &beta, structure were revealed: one large antiparallel &beta, sheet in Lypd6 and Lypd6b, and two &beta, sheets in other proteins. &alpha, Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the &beta, structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal &alpha, helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the &alpha, helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.