The Cationminus signpi Interaction.

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

https://science.sciencemag.org/content/sci/253/5022/872.full.pdf

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.

Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.

Crown ethers: sensors for ions and molecular scaffolds for materials and biological models.

The potential of palladacycles: more than just precatalysts

Investigation of receptor—ligand interactions remains an inexhaustible challenge for chemists and biologists. Structural exploration of biological receptors is the starting point for a better understanding of how they function. Photoaffinity labeling is a biochemical approach to identify and characterize receptors targeting further structural investigations. The primary structure of a receptor protein was typically obtained by reverse genetics after exhaustive purification and sequencing of the N-terminal peptide, which allowed the design of the corresponding oligonucleotide probes. Synthesis of these oligonucleotide probes then led to identification of cDNA clones by hybridization. Following this strategy, several membrane neurotransmitter receptors and constituent polypeptides, present in very small quantities in the central nervous system, were identified and their sequence deduced from the cDNA sequence. Since photoaffinity labeling implies the formation of a covalent bond between a radiolabeled ligand analogue and a receptor binding site, it becomes theoretically possible to isolate and sequence radiolabeled peptides and then synthesize the corresponding oligonucleotide probes. Photoaffinity labeling might avoid the critical solubilization and purification steps of the classical approach. To our knowledge, no such example of primary structure determination based on photoaffinity labeling experiments has been reported. However, the extraordinary developments in gene cloning technologies, in particular homology cloning and expression cloning, have made this approach obsolete and raised the question of new perspectives for photoaffinity labeling technology. In this article we present an update on selected original developments, as well as new challenges for this method. Photoaffinity labeling not only gives access to structural elements but is also a potential tool for the investigation of functional aspects of biological receptors, for example their role in signal transduction mechanisms.

Recent Trends in Photoaffinity Labeling

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent (/sup 3/H)-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The ..cap alpha..-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the ..cap alpha..-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment ..cap alpha.. 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the ..cap alpha..-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the ..cap alpha..-chain that assign the amino-terminal segment ..cap alpha.. 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified (/sup 3/H)DDF-labeled resides, which are conserved in muscle and neuronal ..cap alpha..-chains but not in the other subunits, may be directly involved in agonistmore » binding.« less

Amino acids of the Torpedo marmorata acetylcholine receptor .alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

Combination of a receptor unit with a rigid shaping unit produces a new type of receptor molecules of the cryptand class, hollow macropolycyclic molecules termed speleands, capable of substrate inclusion. Two members of this category of compounds 1 and 2, have been synthesized by connecting in a single step, a macro-cyclic [18]-N3O3 binding unit with a rigid cyclotriveratrylene unit via three bridges. Compound 1 binds the rnethylammonium cation forming both external and internal complexes; for the latter a ‘speleme’ structure, schematically represented by 15, may be proposed.

Speleands. Macropolycyclic receptor cages based on binding and shaping sub-units. Synthesis and properties of macrocycle cyclotriveratrylene combinations. Preliminary communication

Die Untersuchung von Ligand-Rezeptor-Wechselwirkungen ist und bleibt eine Herausforderung fur Chemiker und Biologen. Auf der Grundlage der Strukturaufklarung von biologischen Rezeptoren gelangt man zu einem besseren Verstandnis ihrer Funktionen. Die Methode der Photoaffinitatsmarkierung ist ein biochemischer Ansatz, um Rezeptoren fur weitere Strukturuntersuchungen zu identifizieren und zu charakterisieren. Die Primarstruktur eines Rezeptorproteins wurde typischerweise durch reverse Genetik erhalten; dazu wurde das Protein ausgiebig gereinigt und das N-terminale Peptid sequenziert. Dies ermoglichte das Design entsprechender Oligonucleotidsonden. Deren Synthese fuhrte durch Hybridisierung zur Identifizierung von cDNA-Klonen. Nach dieser Strategie wurden mehrere membranstandige Neurotransmitterrezeptoren und wichtige Polypeptide, die in sehr geringen Mengen im Zentralnervensystem vorkommen, identifiziert und ihre Sequenz aus der cDNA-Sequenz abgeleitet. Da bei der Photoaffinitatsmarkierung eine kovalente Bindung zwischen einem radioaktiv markierten Analogon des Liganden und der Rezeptorbindungsstelle gebildet wird, ist es theoretisch moglich, radioaktiv markierte Peptide zu isolieren und zu sequenzieren sowie dann die entsprechenden Oligonucleotide zu synthetisieren. Im Vergleich zum klassischen Ansatz konnen durch Photoaffinitatsmarkierung moglicherweise die kritischen Solubilisierungs- und Reinigungsschritte vermieden werden. So weit uns bekannt ist, wurde bisher noch kein Beispiel fur eine Primarstrukturbestimmung durch Photoaffinitatsmarkierung veroffentlicht. Die ausergewohnlichen Fortschritte der Klonierungsmethoden, vor allem der Homologie- und Expressionsklonierungen, haben den oben geschilderten Ansatz jedoch vollstandig uberholt und so die Frage nach neuen Perspektiven fur die Photoaffinitatsmarkierung aufgeworfen. In diesem Ubersichtsartikel prasentieren wir eine aktualisierte Zusammenstellung ausgewahlter Entwicklungen und neue Herausforderungen fur diese Methode. Insbesondere eroffnet die Methode der Photoaffinitatsmarkierung nicht nur den Zugang zu Strukturelementen, sondern ist auch dafur geeignet, die Funktionen biologischer Rezeptoren, z. B. ihre Rolle bei der Signaltransduktion, zu untersuchen.

Neue Entwicklungen bei der Photoaffinitätsmarkierung

A photoaffinity ligand of the acetylcholine-binding site predominantly labels the region 179–207 of the α-subunit on native acetylcholine receptor from Torpedo marmorata

Florence Kotzyba-Hibert

Papers

Recent Trends in Photoaffinity Labeling

Amino acids of the Torpedo marmorata acetylcholine receptor .alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

Speleands. Macropolycyclic receptor cages based on binding and shaping sub-units. Synthesis and properties of macrocycle cyclotriveratrylene combinations. Preliminary communication

Neue Entwicklungen bei der Photoaffinitätsmarkierung

A photoaffinity ligand of the acetylcholine-binding site predominantly labels the region 179–207 of the α-subunit on native acetylcholine receptor from Torpedo marmorata