scholarly journals Calmodulin Binding to Connexin 35: Specializations to Function as an Electrical Synapse

2020 ◽  
Vol 21 (17) ◽  
pp. 6346
Author(s):  
Jaya Aseervatham ◽  
Xiaofan Li ◽  
Cheryl K. Mitchell ◽  
Ya-Ping Lin ◽  
Ruth Heidelberger ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Electrical Synapse ◽  
Cytoplasmic Calcium ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Connexin 36 ◽  
Calcium Calmodulin Dependent ◽  
C Terminus ◽  
A Site

Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium.

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1230-1236 ◽  
Author(s):  
Zhong Q. Li ◽  
Weiyi Liu ◽  
Kwang S. Park ◽  
Brue S. Sachais ◽  
Gowthani M. Arepally ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Binding Sites ◽  
Heparin Therapy ◽  
Common Complication ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
The Third ◽  
N Terminus ◽  
A Site

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

scholarly journals The dissociation of avidin–biotin complexes by guanidinium chloride

1972 ◽  
Vol 130 (3) ◽  
pp. 707-711 ◽  
Author(s):  
N. M. Green ◽  
E. J. Toms
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Guanidinium Chloride ◽  
A Site ◽  
Avidin Biotin Complex

Avidin molecules in which a fraction of the four binding sites were occupied by biotin did not dissociate completely in 6.4m-guanidinium chloride. Only unoccupied subunits dissociated. The remainder recombined to form the tetrameric avidin–biotin complex. The rate at which unoccupied subunits were unfolded and dissociated was only decreased by one-half in species in which three of the four binding sites were occupied by biotin. These results can be explained by assuming that unfolding of unoccupied subunits followed by dissociation from the tetramer is initiated by penetration of guanidinium ions into the binding site and disorganization of this region of the subunit. When a site is occupied by biotin this pathway is blocked and the subunit does not unfold. Each subunit behaves independently and is not markedly stabilized when neighbouring subunits are occupied.

scholarly journals Structural basis of adenylyl cyclase 9 activation

2021 ◽  
Author(s):  
Chao Qi ◽  
Pia Lavriha ◽  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Inayathulla Mohammed ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Site ◽  
Adenylyl Cyclase ◽  
Conformational Changes ◽  
Structural Basis ◽  
Structural Studies ◽  
Nucleotide Analogue ◽  
C Terminus ◽  
Membrane Bound ◽  
A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

scholarly journals Identification of fibronectin fragments that bind to carboxy-group-modified proteins

1983 ◽  
Vol 215 (3) ◽  
pp. 613-616 ◽  
Author(s):  
M Vuento ◽  
K Sekiguchi ◽  
M Korkolainen
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Carboxy Group ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Plasma Fibronectin ◽  
C Terminus ◽  
Fibronectin Fragments ◽  
Modified Proteins ◽  
Polypeptide Chains

Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.

scholarly journals Distinct properties of Ca2+–calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

2012 ◽  
Vol 140 (5) ◽  
pp. 541-555 ◽  
Author(s):  
Sze-Yi Lau ◽  
Erik Procko ◽  
Rachelle Gaudet
Keyword(s):  
Binding Site ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Feedback Mechanism ◽  
Sequence Motif ◽  
Recognition Sequence ◽  
Calmodulin Binding ◽  
Negative Feedback Mechanism ◽  
C Terminus ◽  
Wide Range

Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca2+–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca2+–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca2+–CaM, respectively. This structure is similar to canonical Ca2+–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca2+–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca2+–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca2+–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.

scholarly journals Binding of Ca2+ to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies

1994 ◽  
Vol 297 (3) ◽  
pp. 615-624 ◽  
Author(s):  
I M Henderson ◽  
Y M Khan ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Constants ◽  
Tryptophan Fluorescence ◽  
Equilibrium Studies ◽  
High Affinity Binding Site ◽  
Effects Of Ph ◽  
A Site ◽  
Cytoplasmic Side

Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1′Ca2. The E1 conformation of the ATPase initially has one high-affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1′Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1′Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the ‘gating’ site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.

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