scholarly journals The protein-binding pocket of Botulinum neurotoxin B accommodates a preassembled synaptotagmin / ganglioside complex

2021 ◽  
Author(s):  
Jorge Ramirez-Franco ◽  
Fodil Azzaz ◽  
Marion Sangiardi ◽  
G&eacuteraldine Ferracci ◽  
Fahamoe Youssouf ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Transmembrane Domain ◽  
Binding Pocket ◽  
Lipid Binding ◽  
Membrane Topology ◽  
Neuronal Membranes ◽  
Natural Variant ◽  
Synaptotagmin 1 ◽  
Binding Pockets ◽  
Serotype B

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of this therapeutic tool bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside. This interaction allows a lipid-binding loop of BoNT/B to engage in a series of concomitant interactions with the glycone part of GT1b and the transmembrane domain of synaptotagmin. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

scholarly journals Dynamic Control of Ca2+ Binding in the C2 Domains of Synaptotagmin 1

2018 ◽  
Author(s):  
Patrick J. Rock ◽  
Austin G. Meyer ◽  
Chantell S. Evans ◽  
Edwin R. Chapman ◽  
R. Bryan Sutton
Keyword(s):  
Structural Changes ◽  
Dynamic Control ◽  
Point Mutations ◽  
Binding Pocket ◽  
Snare Complex ◽  
C2 Domains ◽  
Domain Interactions ◽  
Synaptotagmin 1 ◽  
Binding Pockets ◽  
Dynamics Simulations

AbstractSynaptotagmin senses fluctuations in the Ca2+ environment of neurons near active zones and transduces a signal to the SNARE complex to initiate exocytosis at the presynaptic terminus. The 3D structures of the two tandem C2 domains of synaptotagmin have been determined to high resolution; however, it is currently unclear how each domain dynamically interacts with Ca2+ at the atomic level. To study the mechanistic consequences of the lethal mutations at the AD3 locus, we introduced tyrosine to asparagine point mutations in both the C2A and C2B domains of synaptotagmin 1, and we have constructed a model that describes the relationship between Ca2+ -binding and the structural changes within each C2 domain. We show that the mobility of loop 3 in the Ca2+ binding pocket increases markedly in C2A, while the mobility of loop 1 changes in C2B with the AD3 mutation. This increase in loop mobility results in an increase in the average volume and variance of the Ca2+ -binding pockets of C2A and C2B. The volume of the unbound Ca2+ -binding pocket in C2A is usually restrained by intra-domain interactions between the tyrosine residue at the AD3 locus and residues on loop 3; however, the AD3 mutation decouples the restraint and results in a larger, more variable Ca2+ -binding pocket in C2A. C2B maintains a more compact Ca2+ -binding pocket; however, its volume also fluctuates significantly with the AD3 mutation. Changes in binding pocket volume that involve more variable Ca2+ binding loops would likely affect Ca2+ affinity in the neurons of the affected organism. Using molecular-dynamics simulations, we show that mutations at the AD3 locus alter the mobility of the Ca2+ -binding loops by removing a key stabilization mechanism that is normally present in C2 domains. The lack of loop stabilization results in a net increase in the volume of the Ca2+ -binding pocket and provides an explanation for the observed lethal phenotype.

scholarly journals Cryo-EM structure of the human histamine H1 receptor/Gq complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ruixue Xia ◽  
Na Wang ◽  
Zhenmei Xu ◽  
Yang Lu ◽  
Jing Song ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Intracellular Loop ◽  
Cryo Electron Microscopy ◽  
Allergy Treatment ◽  
Domain 3 ◽  
Extracellular Side ◽  
Key Residues ◽  
Loop 2

AbstractHistamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of “squash to activate and expand to deactivate”. The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-β junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

scholarly journals The structure and flexibility analysis of the Arabidopsis Synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

2021 ◽  
Author(s):  
Juan Luis Benavente ◽  
Dritan Siliqi ◽  
Lourdes Infantes ◽  
Laura Lagartera ◽  
Alberto Mills ◽  
...  
Keyword(s):  
Cell Function ◽  
Lipid Extraction ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Cellular Environment ◽  
Synaptotagmin 1

Cell function requires the maintenance of membrane lipid homeostasis as changes in cellular environment unbalance this equilibrium. The non-vesicular lipid transfer at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites (CS) is central to restore it. Extended synaptotagmins (E-Syts) are ER proteins that play a central role in this process as they act as molecular tethers with PM and as lipid transfer proteins between these organelles. E-Syts are constitutively anchored to the ER through an N-terminal hydrophobic segment and bind to the PM via C-terminal C2 domains. In plants, synaptotagmins (SYTs) are orthologous of E-Syts and regulate the ER-PM communication by the activity of their two C2 domains in response to abiotic stresses. We have combined macromolecular crystallography, small-angle X-ray scattering, structural bioinformatics and biochemical data to analyze the regulation of plant synaptotagmin 1 (SYT1). Our data show that the binding of SYT1 to the PM is regulated by the interaction of the first C2 domain through a Ca2+-dependent lipid binding site and by a site for phosphorylated forms of phosphatidylinositol in such a way that two different molecular signals are integrated in response to stress. In addition, our data show that SYT1 is highly flexible by virtue of up to three hinge points, including one that connects the two C2 domains. This feature provides conformational freedom to SYT1 to define a large and complementary interaction surface with the PM. This structural plasticity, in turn, may facilitate lipid extraction, protein loading and subsequent transfer between PM and ER.

scholarly journals A Simple, Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection

PLoS ONE ◽  
2014 ◽  
Vol 9 (12) ◽  
pp. e114124 ◽  
Author(s):  
Jiubiao Guo ◽  
Ci Xu ◽  
Xuechen Li ◽  
Sheng Chen
Keyword(s):  
Botulinum Neurotoxin ◽  
Serotype B

scholarly journals Characterization of the anti-apoptotic mechanism of Bcl-B

2003 ◽  
Vol 376 (1) ◽  
pp. 229-236 ◽  
Author(s):  
Dayong ZHAI ◽  
Ning KE ◽  
Haichao ZHANG ◽  
Uri LADROR ◽  
Mary JOSEPH ◽  
...  
Keyword(s):  
Strong Correlation ◽  
Transmembrane Domain ◽  
Binding Pocket ◽  
Over Expression ◽  
Bh3 Domain ◽  
Family Protein ◽  
Critical Residues ◽  
Differential Binding ◽  
Apoptotic Mechanism

Bcl-B protein is an anti-apoptotic member of the Bcl-2 family protein that contains all the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal transmembrane domain. Our previous results showed that Bcl-B binds Bax and suppresses apoptosis induced by over-expression of Bax; however, Bcl-B does not bind or suppress Bak. To explore the molecular basis for the differential binding and suppression of Bax and Bak, we studied the BH3 dimerization domains of Bax and Bak. Chimeric mutants of Bax and Bak were generated that swapped the BH3 domains of these pro-apoptotic proteins. Bcl-B associated with and blocked apoptosis induced by mutant Bak containing the BH3 domain of Bax, but not mutant Bax containing the BH3 domain of Bak. In contrast, Bcl-XL protein bound and suppressed apoptosis induction by Bax, Bak and both BH3-domain chimeras. A strong correlation between binding and apoptosis suppression was also obtained using a series of alanine substitutions spanning the length of the Bax BH3 domain to identify critical residues for Bcl-B binding. Conversely, using structure-based modelling to design mutations in the BH3-binding pocket of Bcl-B, we produced two Bcl-B mutants (Leu86→Ala and Arg96→Gln) that failed to bind Bax and that also were unable to suppress apoptosis induced by Bax over-expression. In contrast, other Bcl-B mutants that still bound Bax retained protective activity against Bax-induced cell death, thus serving as a control. We conclude that, in contrast with some other anti-apoptotic Bcl-2-family proteins, a strong correlation exists for Bcl-B between binding to pro-apoptotic multidomain Bcl-2 family proteins and functional apoptosis suppression.

scholarly journals Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

Toxins ◽  
2015 ◽  
Vol 7 (12) ◽  
pp. 5068-5078 ◽  
Author(s):  
Luisa Cheng ◽  
Thomas Henderson ◽  
Tina Lam ◽  
Larry Stanker
Keyword(s):  
Monoclonal Antibodies ◽  
Botulinum Neurotoxin ◽  
Sensitive Detection ◽  
Serotype B

Fluorescent nanoparticle sensors with tailor-made recognition units and proximate fluorescent reporter groups

2018 ◽  
Vol 42 (12) ◽  
pp. 9377-9380 ◽  
Author(s):  
Xiaoyu Xing ◽  
Yan Zhao
Keyword(s):  
Molecular Imprinting ◽  
Binding Pocket ◽  
Fluorescent Sensors ◽  
Covalent Modification ◽  
Fluorescent Reporter ◽  
Binding Pockets ◽  
Fluorescent Nanoparticle

Molecular imprinting in micelles followed by covalent modification of the binding pocket yielded fluorescent sensors with precisely constructed binding pockets.

scholarly journals Exploring Multi‐Subsite Binding Pockets in Proteins: DEEP‐STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B

2020 ◽  
Vol 26 (44) ◽  
pp. 10024-10034
Author(s):  
Serena Monaco ◽  
Samuel Walpole ◽  
Hassan Doukani ◽  
Ridvan Nepravishta ◽  
Macarena Martínez‐Bailén ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Cholera Toxin ◽  
Binding Pocket ◽  
Toxin B ◽  
Cholera Toxin B ◽  
Binding Pockets ◽  
Std Nmr

scholarly journals Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT3Aserotonin receptor

2019 ◽  
Vol 117 (1) ◽  
pp. 405-414 ◽  
Author(s):  
Nicholas B. Guros ◽  
Arvind Balijepalli ◽  
Jeffery B. Klauda
Keyword(s):  
Serotonin Receptor ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Allosteric Regulation ◽  
Md Simulations ◽  
Binding Pocket ◽  
Careful Examination ◽  
Dynamic Binding ◽  
Order Of Magnitude ◽  
Membrane Bound

Aided by efforts to improve their speed and efficiency, molecular dynamics (MD) simulations provide an increasingly powerful tool to study the structure–function relationship of pentameric ligand-gated ion channels (pLGICs). However, accurate reporting of the channel state and observation of allosteric regulation by agonist binding with MD remains difficult due to the timescales necessary to equilibrate pLGICs from their artificial and crystalized conformation to a more native, membrane-bound conformation in silico. Here, we perform multiple all-atom MD simulations of the homomeric 5-hydroxytryptamine 3A (5-HT3A) serotonin receptor for 15 to 20 μs to demonstrate that such timescales are critical to observe the equilibration of a pLGIC from its crystalized conformation to a membrane-bound conformation. These timescales, which are an order of magnitude longer than any previous simulation of 5-HT3A, allow us to observe the dynamic binding and unbinding of 5-hydroxytryptamine (5-HT) (i.e., serotonin) to the binding pocket located on the extracellular domain (ECD) and allosteric regulation of the transmembrane domain (TMD) from synergistic 5-HT binding. While these timescales are not long enough to observe complete activation of 5-HT3A, the allosteric regulation of ion gating elements by 5-HT binding is indicative of a preactive state, which provides insight into molecular mechanisms that regulate channel activation from a resting state. This mechanistic insight, enabled by microsecond-timescale MD simulations, will allow a careful examination of the regulation of pLGICs at a molecular level, expanding our understanding of their function and elucidating key structural motifs that can be targeted for therapeutic regulation.

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