Cation Diffusion Facilitators Transport Initiation and Regulation Is Mediated by Cation Induced Conformational Changes of the Cytoplasmic Domain

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

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The MscS Cytoplasmic Domain and Its Conformational Changes on the Channel Gating

Mechanosensitive Ion Channels, Part A - Current Topics in Membranes ◽

10.1016/s1063-5823(06)58011-1 ◽

2007 ◽

pp. 295-309 ◽

Cited By ~ 2

Author(s):

Piotr Koprowski ◽

Wojciech Grajkowski ◽

Andrzej Kubalski

Keyword(s):

Conformational Changes ◽

Channel Gating ◽

Cytoplasmic Domain

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A Phosphorylation-Dependent Binding of 14-3-3ζ to the Central Region of the GPIbalpha Cytoplasmic Domain.

Blood ◽

10.1182/blood.v114.22.2995.2995 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2995-2995

Author(s):

Yanhong Yuan ◽

Weilin Zhang ◽

Rong Yan ◽

Yi Liao ◽

Lili Zhao ◽

...

Keyword(s):

Conformational Changes ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Vascular Wall ◽

Membrane Skeleton ◽

Cytoplasmic Domain ◽

Filamin A ◽

Flow Conditions ◽

Binding Function ◽

Truncation Mutant

Abstract Abstract 2995 Poster Board II-973 Introduction: The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX. Methods and Results: A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding and diminished ability to mediate cell adhesion to VWF under flow conditions. Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX. Disclosures: No relevant conflicts of interest to declare.

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Conformational Changes in the Cytoplasmic Domain of theEscherichia coliAspartate Receptor upon Adaptive Methylation†

Biochemistry ◽

10.1021/bi980343y ◽

1998 ◽

Vol 37 (42) ◽

pp. 14852-14859 ◽

Cited By ~ 14

Author(s):

Hervé Le Moual ◽

Tony Quang ◽

Daniel E. Koshland

Keyword(s):

Conformational Changes ◽

Cytoplasmic Domain

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ACTIVATION LOOP PHOSPHORYLATION OF A NON-RD RECEPTOR KINASE INITIATES PLANT INNATE IMMUNE SIGNALING

10.1101/2021.05.01.442257 ◽

2021 ◽

Author(s):

Kyle W Bender ◽

Daniel Couto ◽

Yasuhiro Kadota ◽

Alberto P Macho ◽

Jan Sklenar ◽

...

Keyword(s):

Protein Kinase ◽

Conformational Changes ◽

Stress Responses ◽

Elongation Factor ◽

Cytoplasmic Domain ◽

Dependent Manner ◽

Immune Signaling ◽

Receptor Kinases

Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor which could initiate signaling in a feed-forward fashion either allosterically or through driving the dissociation of negative regulators of the complex.

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Cryo-EM analysis of PIP2 regulation in mammalian GIRK channels

eLife ◽

10.7554/elife.60552 ◽

2020 ◽

Vol 9 ◽

Author(s):

Yiming Niu ◽

Xiao Tao ◽

Kouki K Touhara ◽

Roderick MacKinnon

Keyword(s):

Conformational Changes ◽

Cytoplasmic Membrane ◽

Membrane Surface ◽

Cytoplasmic Domain ◽

Inward Rectifier ◽

Particle Analysis ◽

Single Particle Analysis ◽

Girk Channels ◽

Binding Surface ◽

To Receive

G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP2. Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of neuronal GIRK2 as a function of the C8-PIP2 concentration. We find that PIP2 shifts the equilibrium between two distinguishable structures of neuronal GIRK (GIRK2), extended and docked, towards the docked form. In the docked form the cytoplasmic domain, to which Gβγ binds, becomes accessible to the cytoplasmic membrane surface where Gβγ resides. Furthermore, PIP2 binding reshapes the Gβγ binding surface on the cytoplasmic domain, preparing it to receive Gβγ. We find that cardiac GIRK (GIRK1/4) can also exist in both extended and docked conformations. These findings lead us to conclude that PIP2 influences GIRK channels in a structurally similar manner to Kir2.2 channels. In Kir2.2 channels, the PIP2-induced conformational changes open the pore. In GIRK channels, they prepare the channel for activation by Gβγ.

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From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria

PLoS ONE ◽

10.1371/journal.pone.0231839 ◽

2020 ◽

Vol 15 (4) ◽

pp. e0231839

Author(s):

Noa Keren-Khadmy ◽

Natalie Zeytuni ◽

Nitzan Kutnowski ◽

Guy Perriere ◽

Caroline Monteil ◽

...

Keyword(s):

Cation Diffusion ◽

Cation Diffusion Facilitators

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Evidence from Site-directed Mutagenesis that the Cytoplasmic Domain of the β3 Subunit Influences the Conformational State of the αvβ3 Integrin Ectodomain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615658 ◽

2001 ◽

Vol 85 (04) ◽

pp. 716-723 ◽

Cited By ~ 7

Author(s):

Elisabeth Schaffner-Reckinger ◽

Nicolaas Brons ◽

Nelly Kieffer

Keyword(s):

Conformational Changes ◽

Structural Integrity ◽

Cytoplasmic Domain ◽

Cation Binding ◽

Site Directed Mutagenesis ◽

Metabolic Energy ◽

Minor Effect ◽

Αvβ3 Integrin ◽

Vitronectin Receptor ◽

A Minor

SummaryIn order to explore the mechanisms leading to conformational changes of the vitronectin receptor αvβ3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on 3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire 3 cytoplasmic domain induced constitutive LIBS exposure on αvβ3 and IIb β3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on αvβ3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the β3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the αvβ3 ectodo-main, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes.

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