Palmer Taylor

Structures of Aplysia Achbp Complexes with Nicotinic Agonists and Antagonists Reveal Distinctive Binding Interfaces and Conformations.

Upon ligand binding at the subunit interfaces, the extracellular domain of the nicotinic acetylcholine receptor undergoes conformational changes, and agonist binding allosterically triggers opening of the ion channel. The soluble acetylcholine‐binding protein (AChBP) from snail has been shown to be a structural and functional surrogate of the ligand‐binding domain (LBD) of the receptor. Yet, individual AChBP species display disparate affinities for nicotinic ligands. The crystal structure of AChBP from Aplysia californica in the apo form reveals a more open loop C and distinctive positions for other surface loops, compared with previous structures. Analysis of Aplysia AChBP complexes with nicotinic ligands shows that loop C, which does not significantly change conformation upon binding of the antagonist, methyllycaconitine, further opens to accommodate the peptidic antagonist, α‐conotoxin ImI, but wraps around the agonists lobeline and epibatidine. The structures also reveal extended and nonoverlapping interaction surfaces for the two antagonists, outside the binding loci for agonists. This comprehensive set of structures reflects a dynamic template for delineating further conformational changes of the LBD of the nicotinic receptor.

A Splice Code for trans-Synaptic Cell Adhesion Mediated by Binding of Neuroligin 1 to α- and β-Neurexins

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic β-neurexins, but not with presynaptic α-neurexins. Unexpectedly, we now find that neuroligins also bind α-neurexins and that α- and β-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines α-neurexin binding but leaves β-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only β-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both α- and β-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to α- and β-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.

Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site

The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20–2.35 Å resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non‐catalytic heterologous protein associations.

Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex.

The crystal structure of the snake toxin fasciculin, bound to mouse acetylcholinesterase (mAChE), at 3.2 A resolution reveals a synergistic three-point anchorage consistent with the picomolar dissociation constant of the complex. Loop II of fasciculin contains a cluster of hydrophobic residues that interact with the peripheral anionic site of the enzyme and sterically occlude substrate access to the catalytic site. Loop I fits in a crevice near the lip of the gorge to maximize the surface area of contact of loop II at the gorge entry. The fasciculin core surrounds a protruding loop on the enzyme surface and stabilizes the whole assembly. Upon binding of fasciculin, subtle structural rearrangements of AChE occur that could explain the observed residual catalytic activity of the fasciculin-enzyme complex.

LRRTM2 Interacts with Neurexin1 and Regulates Excitatory Synapse Formation

We identify the leucine-rich repeat transmembrane protein LRRTM2 as a key regulator of excitatory synapse development and function. LRRTM2 localizes to excitatory synapses in transfected hippocampal neurons, and shRNA-mediated knockdown of LRRTM2 leads to a decrease in excitatory synapses without affecting inhibitory synapses. LRRTM2 interacts with PSD-95 and regulates surface expression of AMPA receptors, and lentivirus-mediated knockdown of LRRTM2 in vivo decreases the strength of evoked excitatory synaptic currents. Structure-function studies indicate that LRRTM2 induces presynaptic differentiation via the extracellular LRR domain. We identify Neurexin1 as a receptor for LRRTM2 based on affinity chromatography. LRRTM2 binds to both Neurexin 1alpha and Neurexin 1beta, and shRNA-mediated knockdown of Neurexin1 abrogates LRRTM2-induced presynaptic differentiation. These observations indicate that an LRRTM2-Neurexin1 interaction plays a critical role in regulating excitatory synapse development.

Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors

The crystal structure of the snake long α‐neurotoxin, α‐cobratoxin, bound to the pentameric acetylcholine‐binding protein (AChBP) from Lymnaea stagnalis, was solved from good quality density maps despite a 4.2 Å overall resolution. The structure unambiguously reveals the positions and orientations of all five three‐fingered toxin molecules inserted at the AChBP subunit interfaces and the conformational changes associated with toxin binding. AChBP loops C and F that border the ligand‐binding pocket move markedly from their original positions to wrap around the tips of the toxin first and second fingers and part of its C‐terminus, while rearrangements also occur in the toxin fingers. At the interface of the complex, major interactions involve aromatic and aliphatic side chains within the AChBP binding pocket and, at the buried tip of the toxin second finger, conserved Phe and Arg residues that partially mimic a bound agonist molecule. Hence this structure, in revealing a distinctive and unpredicted conformation of the toxin‐bound AChBP molecule, provides a lead template resembling a resting state conformation of the nicotinic receptor and for understanding selectivity of curaremimetic α‐neurotoxins for the various receptor species.

Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel

Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.

An inositol tetrakisphosphate-containing phospholipid in activated neutrophils

Inositol (l,4,5)trisphosphate (InsP3)1 and tetrakisphosphate (InsP4)2 have been observed in a variety of cell types and have been proposed to play roles in the receptor-mediated rise in intracellular Ca2+ (refs 2, 3). Recently, they have been shown to act synergistically in the activation of a Ca2+-dependent K+ channel in lacrimal acinar cells3. InsP3 is the product of phospholipase C (PLC) action on phosphatidylinositol 4,5-bisphosphate (PtdInsP2), whereas InsP4 is believed to arise from phosphorylation of InsP3 by a cytosolic kinase4. Although sought as a source for InsP4, PtdInsP3 has not been identified in any specific cell type2. There were early reports of InsP4-containing phospholipids in crude extract from bovine brain5, but this finding was later withdrawn6. Recently, however, a membrane-bound enzyme (Type 1 PI kinase) which adds phosphate onto the 3 position of inositol phospholipids has been identified7 and the phosphatidylinositol-3-phosphate (PtdIns(3)P) product characterized. This suggests that several forms of phosphoinositides may exist and could be precursors for some of the variety of soluble inositol phosphate products which have been reported in recent years. Here we report the appearance of another novel phosphoinositide containing four phosphates, phosphatidylinositol trisphosphate (PtdInsP3) which we find only in activated but not in unstimulated neutrophils from human donors.

The Arg451Cys-Neuroligin-3 Mutation Associated with Autism Reveals a Defect in Protein Processing

The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the β-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished β-neurexin-1 (NX1β) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1β. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.

Molecular dissection of subunit interfaces in the acetylcholine receptor: Identification of determinants of α-Conotoxin M1 selectivity

The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.