Philippe Leone

Structural Analysis of the Synaptic Protein Neuroligin and Its β-Neurexin Complex: Determinants for Folding and Cell Adhesion

The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a beta-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an alpha/beta-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism.

Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan.

In Drosophila the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathways. The Toll signaling pathway responds mainly to Gram-positive bacterial and fungal infection while the Imd pathway mediates the response to Gram-negative bacteria. Microbial recognition upstream of Toll involves, at least in part, peptidoglycan recognition proteins (PGRPs). The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and Gram-negative binding protein 1 (GNBP1) that cooperate to detect the presence of lysine-type peptidoglycan in the host. Recently it has been shown that a loss-of-function mutation in peptidoglycan recognition protein SD (PGRP-SD) severely exacerbates the PGRP-SA and GNBP1 mutant phenotypes. Here we have solved the crystal structure of PGRP-SD at 1.5A resolution. Comparison with available structures of PGRPs in complex with their peptidoglycan (PGN) ligand strongly suggests a diaminopimelic acid (DAP) specificity for PGRP-SD. This result is supported by pull-down assays with insoluble PGNs. In addition we show that Toll pathway activation after infection by DAP-type PGN containing bacteria is clearly reduced in PGRP-SD mutant flies. Our hypothesis is that the role of PGRP-SD is the recognition of DAP-type PGNs responsible for the activation of the Toll pathway by Gram-negative bacteria.

The Drosophila Peptidoglycan-Recognition Protein Lf Interacts with Peptidoglycan-Recognition Protein Lc to Downregulate the Imd Pathway.

The peptidoglycan (PGN)‐recognition protein LF (PGRP‐LF) is a specific negative regulator of the immune deficiency (Imd) pathway in Drosophila. We determine the crystal structure of the two PGRP domains constituting the ectodomain of PGRP‐LF at 1.72 and 1.94 Å resolution. The structures show that the LFz and LFw domains do not have a PGN‐docking groove that is found in other PGRP domains, and they cannot directly interact with PGN, as confirmed by biochemical‐binding assays. By using surface plasmon resonance analysis, we show that the PGRP‐LF ectodomain interacts with the PGRP‐LCx ectodomain in the absence and presence of tracheal cytotoxin. Our results suggest a mechanism for downregulation of the Imd pathway on the basis of the competition between PRGP‐LCa and PGRP‐LF to bind to PGRP‐LCx.

Structural insights into the exquisite selectivity of neurexin/neuroligin synaptic interactions.

The extracellular domains of neuroligins and neurexins interact through Ca2+ to form flexible trans‐synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic maturation and differentiation, is regulated by gene selection, alternative mRNA splicing and post‐translational modifications. A new, 2.6 Å‐resolution crystal structure of a soluble neurexin‐1β–neuroligin‐4 (Nrx1β–NL4) complex permits a detailed description of the Ca2+‐coordinated interface and unveils concerted positional rearrangements of several residues of NL4, not observed in neuroligin‐1, associated with Nrx1β binding. Surface plasmon resonance analysis of the binding of structure‐guided Nrx1β mutants towards NL4 and neuroligin‐1 shows that flexibility of the Nrx1β‐binding site in NL4 is reflected in a greater dissociation constant of the complex and higher sensitivity to ionic strength and pH variations. Analysis of neuroligin mutants points to critical functions for two respective residues in neuroligin‐1 and neuroligin‐2 in governing the affinity of the complexes. Although neuroligin‐1 and neuroligin‐2 have pre‐determined conformations that respectively promote and prevent Nrx1β association, unique conformational reshaping of the NL4 surface is required to permit Nrx1β association.

Lead-antimony sulfosalts from Tuscany (Italy). VI. Pellouxite, ∼ (Cu,Ag)2Pb21Sb23S55ClO, a new oxy-chloro-sulfosalt from Buca della Vena mine, Apuan Alps

Pellouxite, the second natural oxy-chloro-sulfosalt after pillaite, has been discovered in cavities of calcite veins in the small Fe-Ba deposit of Buca della Vena mine, Apuan Alps (Tuscany, Italy). The new mineral name honours professor Alberto Pelloux (1868–1948), curator of the mineralogical Museum at the University of Genova. Pellouxite occurs as black acicular flattened crystals with metallic lustre, up to 1 mm long and less than 0.05 mm thick. Main reflectance values [λ (nm), R air / R oil %]: 470, 38.3/23.8; 546, 37.3/22.4; 589, 36.9/21.5; 650, 35.2/19.6. It has monoclinic symmetry, space group C 2/ m , with a = 55.824(11), b = 4.0892(8), c = 24.128(5) A, β = 113.14(3)° and V = 5065(2) A 3 . The eight strongest lines of the X-ray powder diffraction pattern are [d(A), I obs (hkl)]: 4.002, 38 (606, 406); 3.878, 24 (206, 112); 3.562, 31 (804, 1202); 3.423, 100 (807, 1604, 407); 3.009, 25 (808, 912, 713); 2.948, 27 (1313, 1111); 2.265, 19 (409); 2.048, 20 (020). Electron probe microanalysis gives (mean of 15 spots; wt.%, error σ): Pb 47.17(20), Sb 31.16(23), Cu 0.89(5), Ag 0.59(5), S 19.08(6), Cl 0.33(3), O 0.39(15), Total 99.60. The unit formula, on the basis of Pb + Sb = 22 at. according to the crystal structure study, is (Cu 0.64 Ag 0.25 )Pb 10.36 Sb 11.64 S 27.07 Cl 0.42 O 1.11 ( Z = 4). Taking into account a 2 b superstructure, the crystal chemical formula is (Cu,Ag) 2-x Pb 21-x Sb 23+x S 55 ClO (x = 0.12; d calc. = 5.97 g/cm 3 ), derived from the stoichiometric one (Cu,Ag) 2 Pb 21 Sb 23 S 55 ClO. Pellouxite, structurally related to scainiite, is an expanded monoclinic derivative of synthetic hexagonal Ba 12 Bi 24 S 48 . It belongs to the zinkenite group of lead sulfosalts with cyclically twinned structures and their derivatives. Like for pillaite, its formation is the result of a complex sulfide deposition from hydrothermal brines, at relatively high temperature, and fO 2 /fS 2 conditions controlled by the pyrite-hematite-magnetite buffer.

X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form

Background: Monalysin is a β-barrel pore-forming toxin secreted by Pseudomonas entomophila. Results: Monalysin structure belongs to the Aerolysin fold family but is devoid of receptor-binding domain; pro-Monalysin forms a stable 18-mer complex. Conclusion: The delivery of 18 subunits may bypass the requirement of receptor-dependent concentration. Significance: The functional mechanism of Monalysin differs from that of described Aerolysin family members. β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.

Synthesis and structural studies of manganese oxyhalides with a multisite framework: Part two: Mn7.5O10Br3, a new lacunar oxybromide

Abstract MnBr2 reacts with MnO2 in a temperature range of 300 – 500°C to produce a new manganese oxybromide. Chemical analysis and structural determination indicate that the oxybromide possesses a chemical formula, Mn7.5O10Br3, which is different from the manganese oxychloride. The symmetry is tetragonal with a = 9.327(5) Ȧ. and c = 13.104 (3) A . The structure has been solved in the space group 14/mmm with Z = 4, R = 0.046. The oxybromide appears as a lacunar equivalent of Mn8O10Cl3 with empty Br6 octahedra.

Structure-function relationships of the α/β-hydrolase fold domain of neuroligin: A comparison with acetylcholinesterase

The neuroligins are postsynaptic cell adhesion proteins whose extracellular domain belongs to the alpha/beta-hydrolase fold family of proteins, a family characterized through the enzyme acetylcholinesterase (AChE) and other enzymes with various substrate specificities. Neuroligin associations with the pre-synaptic neurexins participate in synapse maturation and maintenance. Alternative splicing of the neuroligin and neurexin genes results in multiple isoforms and presumably regulation of activity, while mutations appear to be associated with autism spectrum disorders. The crystal structures of the extracellular, cell adhesion domain of three neuroligins (NL1, NL2 and NL4) revealed features that distinguish the neuroligins from their enzyme relatives and could not be predicted by homology modelling from an AChE template. The structures of NL1 and NL4 bound with a soluble beta-neurexin domain (Nrxbeta1) revealed the precise position and orientation of the bound Nrxbeta1 and the Ca(2+)-dependent interaction network at the complex interface. Herein we present an overview of the unbound and Nrxbeta1-bound neuroligin structures and compare them with structures of AChEs with and without a bound fasciculin partner. This study exemplifies how an alpha/beta-hydrolase fold domain tailored for catalysis varies to acquire adhesion properties, and defines three surface regions with distinctive locations and properties for homologous or heterologous partner association.

Synthesis and structural studies of manganese oxyhalides with a multisite framework: Part one: Mn8O10Cl3

Abstract A redox reaction between MnO2 and MnCl2 occurs above 300° C in evacuated sealed tubes yielding the manganese oxychloride, Mn8O10Cl3. This compound is tetragonal with a = 9.255(3) A , and c = 13.068(4) A . The structural determination has been solved in the space group I4/mmm with Z = 4. (R = 0.029). Manganese 2+ are located in two symmetric sites, oxide cubes and chloride octahedra. Mn3+ ions occupy three octahedral sites, i.e. one undistorted oxygen octahedron and two highly distorted oxychloride octahedra. The chemical formulation is therefore better described as: Mn2+Mn3+7O10Cl3.

Investigations by magnetic measurements and neutron diffraction studies of the magnetic ordering of Mn8O10C13

Abstract The three dimensional manganese oxychloride Mn 8 O 10 C1 3 belongs to the quadratic symmetry, space group 14/mmm with cell parameters: a = 9.275(2) rmA and c = 13.057(2)A, ( Z = 4) and orders magnetically below 29 K. The susceptibility versus temperature curve suggests a nonclassical ferrimagnetic behavior. Above 200 K, manganese oxychloride follows a classical Curie-Weiss law. Spontaneous magnetization increases from 5 K, reaching a maximum at 20 K and decreases to zero at 30 K. A neutron diffraction study has been performed between 2 and 100 K. The magnetic structure is built on two magnetic sublattices. One is ferrimagnetic collinear with the a axis, and the second one is frustrated. Magnetization behavior can be explained in terms of orientation and intensity changes in magnetic moments carried by manganese atoms.