Using Liquid Crystals to Amplify Protein−Receptor Interactions:  Design of Surfaces with Nanometer-Scale Topography that Present Histidine-Tagged Protein Receptors†

Langmuir ◽  
2003 ◽  
Vol 19 (5) ◽  
pp. 1671-1680 ◽  
Author(s):  
Yan-Yeung Luk ◽  
Matthew L. Tingey ◽  
David J. Hall ◽  
Barbara A. Israel ◽  
Christopher J. Murphy ◽  
...  
Keyword(s):  
Liquid Crystals ◽  
Nanometer Scale ◽  
Protein Receptor ◽  
Receptor Interactions ◽  
Protein Receptors

scholarly journals Structural and Biochemical Characterization of Botulinum Neurotoxin Subtype B2 Binding to Its Receptors

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 603
Author(s):  
Jonathan R. Davies ◽  
Geoffrey Masuyer ◽  
Pål Stenmark
Keyword(s):  
Receptor Binding ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Hydrophobic Interactions ◽  
Binding Assays ◽  
Protein Receptor ◽  
Wide Range ◽  
Protein Receptors ◽  
Therapeutic Molecules ◽  
Botulinum Neurotoxins

Botulinum neurotoxins (BoNTs) can be used therapeutically to treat a wide range of neuromuscular and neurological conditions. A collection of natural BoNT variants exists which can be classified into serologically distinct serotypes (BoNT/B), and further divided into subtypes (BoNT/B1, B2, …). BoNT subtypes share a high degree of sequence identity within the same serotype yet can display large variation in toxicity. One such example is BoNT/B2, which was isolated from Clostridium botulinum strain 111 in a clinical case of botulism, and presents a 10-fold lower toxicity than BoNT/B1. In an effort to understand the molecular mechanisms behind this difference in potency, we here present the crystal structures of BoNT/B2 in complex with the ganglioside receptor GD1a, and with the human synaptotagmin I protein receptor. We show, using receptor-binding assays, that BoNT/B2 has a slightly higher affinity for GD1a than BoNT/B1, and confirm its considerably weaker affinity for its protein receptors. Although the overall receptor-binding mechanism is conserved for both receptors, structural analysis suggests the lower affinity of BoNT/B2 is the result of key substitutions, where hydrophobic interactions important for synaptotagmin-binding are replaced by polar residues. This study provides a template to drive the development of future BoNT therapeutic molecules centered on assessing the natural subtype variations in receptor-binding that appears to be one of the principal stages driving toxicity.

scholarly journals Syntaxin 7 and VAMP-7 are SolubleN-Ethylmaleimide–sensitive Factor Attachment Protein Receptors Required for Late Endosome–Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages

2000 ◽  
Vol 11 (7) ◽  
pp. 2327-2333 ◽  
Author(s):  
Diane McVey Ward ◽  
Jonathan Pevsner ◽  
Matthew A. Scullion ◽  
Michael Vaughn ◽  
Jerry Kaplan
Keyword(s):  
Alveolar Macrophages ◽  
Transmembrane Domain ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Protein Receptors

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome–lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome–lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome–lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome–lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

Controlling Materials Architecture on the Nanometer-Scale: PPV Nanocomposites Via Polymerizable Lyotropic Liquid Crystals

1997 ◽  
Vol 488 ◽  
Author(s):  
Ryan C. Smith ◽  
Hai Deng ◽  
Walter M. Fischer ◽  
Douglas L. Gin
Keyword(s):  
Liquid Crystals ◽  
Solid State ◽  
Liquid Crystalline ◽  
Lyotropic Liquid Crystals ◽  
Nanometer Scale ◽  
General Strategy ◽  
Hexagonal Symmetry ◽  
The Matrix ◽  
Hexagonal Arrangement ◽  
Lock In

AbstractWe have developed a general strategy for the construction of ordered nanocomposites with hexagonal symmetry, using polymerizable lyotropic (i.e., amphiphilic) liquid crystals. In this approach, self-organizing lyotropic liquid-crystalline monomers are used to form an ordered template matrix in the presence of a reactive hydrophilic solution. Subsequent photopolymerization to lock-in the matrix architecture, followed by initiation of chemistry within the ordered hydrophilic domains to afford solid-state fillers, yields the anisotropic nanocomposites. Composites have been synthesized that have a regular hexagonal arrangement of extended poly(p-phenylenevinylene) (PPV) domains, with a regular interchannel spacing of 4 nm. The photoluminescence of these materials is significantly altered from that of bulk PPV. The dimensions of these nanocomposites can be tuned by varying the size of the hydrophobic tails and/or the nature of the counterion associated with the hydrophilic headgroup of the monomer.

scholarly journals C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor

2000 ◽  
Vol 347 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Judit HERREROS ◽  
Giovanna LALLI ◽  
Giampietro SCHIAVO
Keyword(s):  
Cell Surface ◽  
Structural Information ◽  
Neuronal Cell ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Protein Toxin ◽  
Protein Receptor ◽  
Protein Receptors ◽  
Neuronal Cell Lines ◽  
Additional Protein

Tetanus neurotoxin (TeNT) is a powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, an essential protein of the synaptic vesicle fusion machinery, and consequently blocks neurotransmission. The extreme neurospecificity of TeNT is determined by the binding of its C-terminal domain (fragment C or HC) to neuronal receptors. Whereas polysialogangliosides are known acceptors for the toxin, the existence of additional protein receptors has also been suggested. We have reported previously on a 15 kDa cell-surface glycoprotein that interacts with TeNT in neuronal cell lines and motoneurons [Herreros, Lalli, Montecucco and Schiavo (2000) J. Neurochem., in the press]. Here, on the basis of the structural information provided by the crystallization of fragment C of TeNT, we have expressed its C-and N-terminal halves as recombinant proteins and analysed their binding abilities to rat phaeochromocytoma (PC12) cells differentiated with nerve growth factor. We found that the C-terminal subdomain of the fragment C of TeNT is necessary and sufficient for cell binding and for the interaction with the 15 kDa putative receptor. In contrast, the N-terminal half showed a very poor interaction with the cell surface. These results restrict the binding domain of TeNT to the C-terminal half of the fragment C and highlight the importance of this domain for the neurospecific interaction of the toxin with the synapse. Furthermore, these findings support the use of this portion of TeNT as a neurospecific targeting device, pointing to an involvement of the N-terminal subdomain in later steps of the intoxication pathway.

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