Innokenty V. Maslennikov

Unique dimeric structure of BNip3 transmembrane domain suggests membrane permeabilization as a cell death trigger.

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

NMR spatial structure of α-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors1

A 600 MHz NMR study of α‐conotoxin ImI from Conus imperialis, targeting the α7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin‐spin coupling constants, and amide protons hydrogen‐deuterium exchange data: rmsd of the backbone atom coordinates at the 2–12 region is 0.28 Å in the 20 best structures. The structure is described as a type I β‐turn (positions 2–5) followed bya distorted helix (positions 5–11). Similar structural psattern can be found in all neuronal‐specific α‐conotoxins. Highly mobile side chains of the Asp‐5, Arg‐7 and Trp‐10 residues form a single site for ImI binding to the α7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.

Dynamic conformational changes of extracellular S5–P linkers in the hERG channel

The hERG channel has an unusually long ‘S5–P linker’ (residues 571–613) that lines the outer mouth of the pore. Previously, we have shown that residues along this S5–P linker are critical for the fast‐inactivation process and K+ selectivity of the hERG channel. Here we used several approaches to probe the structure of this S5–P linker and its interactions with other domains of the hERG channel. Circular dichroism and NMR analysis of a synthetic hERG S5–P linker peptide suggested that this linker is quite dynamic: its central region (positions 583–593) can be unstructured or helical, depending on whether it is immersed in an aqueous phase or in contact with a hydrophobic environment. Cysteine introduced into positions 583–597 of the S5–P linker can form intersubunit disulphide bonds, and at least four of them (at 584, 585, 588 and 589) can form disulphide bonds with counterparts from neighbouring subunits. We propose that the four S5–P linkers in a hERG channel can engage in dynamic conformational changes during channel gating, and interactions between S5–P linkers from neighbouring subunits contribute importantly to channel inactivation.

First tryptophan-containing weak neurotoxin from cobra venom.

With the purpose of studying structure-function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seylers Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated alpha-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.

Spatial structure of the M2 transmembrane segment of the nicotinic acetylcholine receptor α‐subunit

A synthetic peptide corresponding to the transmembrane segment M2 (residues 236–267) of the α‐subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by two dimensional 1H‐NMR spectroscopy in a chloroform‐methanol (1:1) mixture containing 0.1 M LiClO4. Reconstruction of the spatial structure of M2 from the NMR data resulted in an α‐helix formed by residues 241–263. Distribution of the molecular hydrophobicity potential on the helix surface is very similar to that in five‐helix bundles of proteins with a known three dimensional structure: two hydrophilic bands located on the opposite helix sides separated by strong hydrophobic zones.

α-Conotoxin GI benzoylphenylalanine derivatives

alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.α‐Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)‐competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in α‐conotoxin binding by use of photoactivated cross‐linking, two benzoylphenylalanine (Bpa) analogs of α‐conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their 1H‐NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced 125I‐labeled GI from the Torpedo californica nAChR. 125I‐labeled GI(Bpa12) bound to two sites on the receptor (Kd 13 and 1800 nm), and on UV irradiation specifically photolabeled the α, γ and δ subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the α subunit, cobratoxin‐inhibited incorporation was limited to the 22‐kDa fragment beginning at αSer173 and containing the agonist‐binding site segment C. In the γ subunit, radioactivity was localized to two distinct peptides containing agonist‐binding site segments F and D: nonglycosylated 24‐kDa and glycosylated 13‐kDa fragments starting at γAla167 and γAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo‐electron microscopy structure of Torpedo marmorata nAChR and X‐ray crystal structures of snail acetylcholine‐binding protein complexes with agonists and antagonists.

A Comparative Study on Selectivity of α-Conotoxins GI and ImI Using Their Synthetic Analogues and Derivatives

Comparative structure-function studies have been carried out for α-conotoxin GI acting on nicotinic acetylcholine receptors (AChR) from mammalian muscles and from the electric organ of the Torpedo californica ray and for α-conotoxin ImI, which targets the neuronal a7 AChR. A series of analogs has been prepared for this purpose: chemically modified derivatives, including a covalently linked dimer of GI, as well as analogs wherein one or several amino acid residues have been changed using solid-phase peptide synthesis. The activity of all compounds was assessed in competition with radioiodinated and/or tritiated α-conotoxin GI for binding to the membrane-bound AChR of Torpedo californica. Binding of radioiodinated α-conotoxin GI dimer was also monitored directly, revealing the largest, as compared to all other analogues, difference in the affinity between the two binding sites in the receptor (KD ∼ 11 and 1200 nM). Comparison of binding data with the results of CD measurements point to important role of the spatial organization of the α-conotoxin second loop in manifestation of their “muscle” or “neuronal” specificity.