scholarly journals Identification of 3-hydroxy-1,2-dimethylpyridine-4(1H)-thione as a metal-binding motif for the inhibition of botulinum neurotoxin A

2020 ◽  
Author(s):  
Lucy Lin ◽  
Lewis D. Turner ◽  
Peter Šilhár ◽  
Sabine Pellett ◽  
Eric A. Johnson ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Light Chain ◽  
Metal Binding ◽  
Botulinum Neurotoxin ◽  
Chain Structure ◽  
Binding Motif ◽  
Botulinum Neurotoxin A ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Metal Binding Motif

The 3,4-HOPTO pharmacophore was identified as an inhibitor of botulinum neurotoxin A light chain. Structure-activity relationships were explored, resulting in a compound that protects SNAP-25 cleavage in motor neurons.

scholarly journals Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

mAbs ◽  
2014 ◽  
Vol 6 (2) ◽  
pp. 446-459 ◽  
Author(s):  
Sebastian Miethe ◽  
Christine Rasetti-Escargueil ◽  
Yvonne Liu ◽  
Siham Chahboun ◽  
Thibaut Pelat ◽  
...  
Keyword(s):  
Light Chain ◽  
Botulinum Neurotoxin ◽  
Botulinum Neurotoxin A ◽  
Immune Library

scholarly journals Metal Binding Motif in the Active Site of the HDV Ribozyme Binds Divalent and Monovalent Ions

Biochemistry ◽  
2011 ◽  
Vol 50 (13) ◽  
pp. 2672-2682 ◽  
Author(s):  
Narayanan Veeraraghavan ◽  
Abir Ganguly ◽  
Jui-Hui Chen ◽  
Philip C. Bevilacqua ◽  
Sharon Hammes-Schiffer ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Binding Motif ◽  
Monovalent Ions ◽  
Hdv Ribozyme ◽  
Metal Binding Motif

scholarly journals Metal ion recognition of the metal-binding motif in hammerhead ribozymes

2003 ◽  
Vol 43 (supplement) ◽  
pp. S28
Author(s):  
Y. Tanaka ◽  
Y. Kasai ◽  
C. Kojima ◽  
K. Yamasaki ◽  
H. Morita ◽  
...  
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Binding Motif ◽  
Hammerhead Ribozymes ◽  
Ion Recognition ◽  
Metal Binding Motif

scholarly journals Biofunctionalized Gold Nanoparticles for Colorimetric Sensing of Botulinum Neurotoxin A Light Chain

2014 ◽  
Vol 86 (5) ◽  
pp. 2345-2352 ◽  
Author(s):  
Xiaohu Liu ◽  
Yi Wang ◽  
Peng Chen ◽  
Yusong Wang ◽  
Jinling Zhang ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Light Chain ◽  
Botulinum Neurotoxin ◽  
Colorimetric Sensing ◽  
Botulinum Neurotoxin A

scholarly journals Assessing Structure-Activity Relationships in FVIII By Integration of Structural, Biophysical, and Biochemical Data Obtained with Anti-FVIII Antibodies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4223-4223 ◽  
Author(s):  
Zhiqian Lucy Liu ◽  
George Bou-Assaf ◽  
Ming Zhang ◽  
Allison Goodman ◽  
Po-lin Chiu ◽  
...  
Keyword(s):  
Human Plasma ◽  
Light Chain ◽  
Inhibitory Activity ◽  
C2 Domain ◽  
Structure Activity Relationships ◽  
Deuterium Uptake ◽  
Negative Stain ◽  
Structure Activity ◽  
Interaction Map ◽  
Hdx Ms

Abstract Introduction Anti-factor VIII (FVIII) antibodies are valuable probes for evaluating structure-activity relationships in FVIII. In this study, we structurally and functionally characterized a panel of anti-FVIII antibodies comprising 36 mouse monoclonal antibodies and 1 camelid nanobody with specificities that span all 5 structural domains of FVIII. Integration of these data into a comprehensive functional interaction map reveals regions that are essential for the function of FVIII and provides a useful guide for researchers engaged in the engineering, quantitation, and purification of FVIII and variants thereof. Materials and Methods Monoclonal mouse IgGs were raised by conventional hybridoma methods or obtained from commercial sources (Green Mountain Antibodies, American Diagnostica). Recombinant B domain-deleted human FVIII (BDD-rFVIII), consisting of a mixture of proteolytically processed forms and dual-chain forms, was generated by transfection of HEK293 cells and purified by immuno-affinity chromatography to >95% purity. Human plasma-derived von Willebrand factor (VWF) was purchased from Haematologic Technologies. The chain and domain specificities of each antibody and competition with VWF for FVIII binding were evaluated by biolayer interferometry (BLI; ForteBio Octet). Pairwise epitope overlap analysis (binning) was performed by both BLI and multiplexed surface plasmon resonance (SPR; BioRad ProteOn). The affinity of each antibody for FVIII was determined by SPR (ProteOn, Biacore T200), and the inhibitory activity of each antibody was determined by the standard Bethesda assay using normal human plasma. The ability of antibodies to inhibit the thrombin-mediated cleavage of FVIII was determined by resolving samples from a digestion time-course on SDS-PAGE and subsequent quantitation by gel densitometry. The morphology of FVIII-Fab fragment complexes was determined by negative stain electron microscopy (EM) with images classified into 25 groups. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to localize antibody epitopes on FVIII by evaluating deuterium uptake in the presence and absence of selected antibodies. Differential deuterium uptake over four hours was evaluated for over 500 unique FVIII-derived peptides that resulted from cleavage with pepsin, with greater than 95% sequence coverage of BDD-FVIII. Results and Conclusions Antibodies in this panel were characterized with respect to FVIII chain and domain specificity. Fifteen antibodies were mapped to the FVIII heavy chain (3 in the A1 domain, 12 in the A2 domain), and 22 antibodies were mapped to the FVIII light chain (8 in A3/C1 domains, 14 in C2 domain). Affinities for FVIII varied widely, with KD values ranging from ~10 nM to ~10 pM. Bethesda inhibitory activities also varied widely, from undetectable levels to greater than 40,000 Bethesda units (BU) per milligram. No correlation was observed between antibody affinity and inhibitory activity, but inhibitory activity was strongly associated with distinct clusters of antibodies having overlapping epitopes. Nine antibodies competed with VWF for binding to FVIII (2 in C1 domain, 7 in C2 domain), and these antibodies were distributed among 3 distinct epitope clusters, indicating that the VWF binding interface is broadly distributed across the C1 and C2 domains. Three C2 domain-specific antibodies that compete with VWF for binding to FVIII also interfered with the liberation of the acidic a3 peptide by thrombin in the absence of VWF. Thrombin-mediated cleavage at other sites in FVIII was not affected by these antibodies, suggesting the presence of a thrombin binding site within the C2 domain that is required specifically for cleavage by thrombin at the N-terminus of the FVIII light chain. The structures of FVIII in complex with 15 individual antibody Fab fragments were determined by negative stain EM for both inhibitory and non-inhibitory antibodies, and precise epitope mapping of selected antibodies was achieved by HDX-MS. Collectively, these data were used to generate an FVIII-antibody interaction map that integrates activity data with structural data spanning the atomic level to the gross morphological level. This integrated map can serve as a resource for the evaluation of structure-activity relationships in FVIII. Disclosures Liu: Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Zhang:Biogen Idec: Employment. Goodman:Biogen Idec: Employment. Walz:Biogen Idec: Honoraria, Research Funding. Peters:Biogen Idec: Employment. Kulman:Biogen Idec: Employment.

scholarly journals Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

2004 ◽  
Vol 166 (7) ◽  
pp. 1003-1014 ◽  
Author(s):  
Gideon Lansbergen ◽  
Yulia Komarova ◽  
Mauro Modesti ◽  
Claire Wyman ◽  
Casper C. Hoogenraad ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Intramolecular Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Binding Motif ◽  
Force Microscopy ◽  
Scanning Force ◽  
Metal Binding Motif

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

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