A new mechanism in the binding between Homer3 EVH1 domain and inositol 1,4,5 trisphosphate receptor suppressor domain

2014 ◽  
Vol 92 (3) ◽  
pp. 163-171
Author(s):  
He Wen ◽  
Hyuk Nam Kwon ◽  
Sunghyouk Park
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Large Scale ◽  
Critical Role ◽  
Binding Assays ◽  
Binding Characteristics ◽  
Binding Partners ◽  
Inositol 1,4,5 Trisphosphate ◽  
Target Binding ◽  
Trisphosphate Receptor

The suppressor domain of inositol 1,4,5 trisphosphate receptor (IP3R) has critical roles in regulating the calcium channel by interacting with many binding partners. The residue 49–53 (PPKKF) of the suppressor domain was suggested to be a canonical Homer EVH1 domain binding site and is also the first a part of calmodulin (CaM) binding site. As CaM-binding of the suppressor domain has been shown to involve large-scale conformational changes, we studied the binding characteristics of the Homer EVH1-suppressor domain with NMR spectroscopy and biochemical pull-down assays for mutants. Our data show that the suppressor domain employs the PPKKF motif in a similar but subtly different way compared to previously characterized interactions, and that the suppressor domain does not undergo large-scale conformational changes. Chemical shift assignments of the Homer3 EVH1 domain found that a new set of residues, located at the opposite side of the previously reported binding site, is also involved in binding, which was confirmed by mutant binding assays. Further analysis suggests that F40 in the new binding sites may have a critical role as a conformational lock-switch in Homer-target binding. The proposed mechanism is implicated in the signaling network involving calcium channels.

scholarly journals Two-state Conformational Changes in Inositol 1,4,5-Trisphosphate Receptor Regulated by Calcium

2002 ◽  
Vol 277 (24) ◽  
pp. 21115-21118 ◽  
Author(s):  
Kozo Hamada ◽  
Tomoko Miyata ◽  
Kouta Mayanagi ◽  
Junji Hirota ◽  
Katsuhiko Mikoshiba
Keyword(s):  
Conformational Changes ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

Maturation of inositol 1,4,5-trisphosphate receptor binding in rabbit tracheal smooth muscle

1992 ◽  
Vol 263 (4) ◽  
pp. L501-L505 ◽  
Author(s):  
C. M. Schramm ◽  
S. T. Chuang ◽  
M. M. Grunstein
Keyword(s):  
Smooth Muscle ◽  
Receptor Binding ◽  
Tracheal Smooth Muscle ◽  
Binding Characteristics ◽  
Adult Tissues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Hill Coefficients ◽  
Trisphosphate Receptor

The mechanisms underlying maturational changes in agonist-mediated airway contractility remain to be identified. Since the signal transduction process coupled to airway contraction involves the Ca(2+)-mobilizing action of the second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], the present study examined 1) whether Ins(1,4,5)P3 binding to its intracellular receptor varies with age in rabbit tracheal smooth muscle (TSM), and 2) whether Ca2+ exerts a modulatory effect on Ins(1,4,5)P3 receptor binding that is age dependent. [3H]Ins(1,4,5)P3 binding was assayed in crude TSM membrane preparations isolated from 2-week-old and adult rabbits. Monophasic Scatchard plots were obtained, reflecting a single binding site, with Hill coefficients of 0.988–0.996. The mean +/- SE values for receptor density (Bmax) and binding affinity [i.e., the dissociation constant (Kd)] were similar in the adult and immature tissues, wherein Bmax = 211 +/- 6 238 +/- 60 fmol/mg protein, respectively; and Kd = 14.1 +/- 0.2 and 11.6 +/- 1.2 nM, respectively. Addition of Ca2+ (10(-8)-10(-3) M) significantly modulated Ins(1,4,5)P3 binding, with opposing maturational effects. In adult tissues, Ca2+ produced dose-dependent inhibition of Ins(1,4,5)P3 binding to 59.5% control, whereas Ins(1,4,5)P3 binding in response to Ca2+ was significantly enhanced in the 2-week-old tissues to 183% control. Collectively, these observations demonstrate that the inherent basal binding characteristics of the Ins(1,4,5)P3 receptor are similar in maturing TSM, but that Ca2+ exerts opposite modulatory actions on Ins(1,4,5)P3 receptor binding in immature and adult tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms

2000 ◽  
Vol 346 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Sara VANLINGEN ◽  
Henk SIPMA ◽  
Patrick DE SMET ◽  
Geert CALLEWAERT ◽  
Ludwig MISSIAEN ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Recombinant Proteins ◽  
Ip3 Receptor ◽  
Binding Characteristics ◽  
Binding Domains ◽  
Receptor Isoforms ◽  
Inositol 1,4,5 Trisphosphate ◽  
Adenophostin A ◽  
Trisphosphate Receptor

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP3R1), IP3R2 and IP3R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP3-binding domain and bound IP3 and adenophostin A with high affinity. Ca2+ and calmodulin were previously found to maximally inhibit IP3 binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC50 of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca2+ inhibited IP3 binding to lbs-3 with an IC50 of approx. 700 nM (37±4% inhibition at 5 μM Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP3 binding to lbs-3 by 37±4%, while IP3 binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP3 binding to lbs-3 by calmodulin was dose-dependent (IC50≈ 2 μM). We conclude that the IP3-binding domains of the various IP3R isoforms differ in binding characteristics for IP3 and adenophostin A, and are differentially modulated by Ca2+ and calmodulin, suggesting that the various IP3R isoforms can have different intracellular functions.

scholarly journals Cooperative Formation of the Ligand-binding Site of the Inositol 1,4,5-Trisphosphate Receptor by Two Separable Domains

1999 ◽  
Vol 274 (1) ◽  
pp. 328-334 ◽  
Author(s):  
Fumio Yoshikawa ◽  
Hirohide Iwasaki ◽  
Takayuki Michikawa ◽  
Teiichi Furuichi ◽  
Katsuhiko Mikoshiba
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Ligand Binding Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

scholarly journals Mutational Analysis of the Ligand Binding Site of the Inositol 1,4,5-Trisphosphate Receptor

1996 ◽  
Vol 271 (30) ◽  
pp. 18277-18284 ◽  
Author(s):  
Fumio Yoshikawa ◽  
Mitsuhiro Morita ◽  
Toshiaki Monkawa ◽  
Takayuki Michikawa ◽  
Teiichi Furuichi ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Mutational Analysis ◽  
Ligand Binding Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

scholarly journals Cryo-EM reveals local and global structural rearrangements in RYR mutants

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Kavita A. Iyer ◽  
Yifan Hu ◽  
Thomas Klose ◽  
Takashi Murayama ◽  
Montserrat Samsó
Keyword(s):  
Long Range ◽  
Conformational Changes ◽  
Large Scale ◽  
Single Point ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
Point Mutations ◽  
Polymorphic Ventricular Tachycardia ◽  
Functional Studies ◽  
Single Point Mutations

Single-point mutations in ryanodine receptors (RYRs), large intracellular Ca2+ channels that play a critical role in EC coupling, are linked to debilitating and lethal disorders such as central core disease, malignant hyperthermia (for the skeletal isoform, RYR1), catecholaminergic polymorphic ventricular tachycardia, and ARVD2 (for the cardiac isoform, RYR2). Mutant RYRs result in elevated [Ca2+]cyto due to steady leak from the sarcoplasmic reticulum. To explore the nature of long-range allosteric mechanisms of malfunction, we determined the structure of two N-terminal domain mutants of RYR1, situated far away from the pore. Cryo-electron microscopy of the N-terminal subdomain A (NTDA) and subdomain C (NTDC) full-length mutants, RYR1 R163C (determined to 3.5 Å resolution), and RYR1 Y522S (determined to 4.0 Å resolution), respectively, reveal large-scale conformational changes in the cytoplasmic assembly under closed-state conditions (i.e., absence of activating Ca2+). The multidomain changes suggest that the mutations induce a preactivated state of the channel in R164C by altering the NTDA+/CD interface, and in Y522S by rearrangement of the α-helical bundle in NTDC. However, the extent of preactivation is considerably higher in Y522S as compared with R163C, which agrees with the increased severity of the Y522S mutation as established by various functional studies. The Y522S mutation represents loss of a spacer residue that is crucial for maintaining optimal orientation of α helices in NTDC, alteration of which has long-range effects felt as far away as ∼100 Å. Additionally, the structure of the Y522S mutant channel under open-state conditions also differs from RYR1 WT open channels. Our developing work with RYR mutants exhibits the diverse mechanisms by which these single-point mutations exert an effect on the channel’s function and highlight the complexity of the multidomain channel, as well as the need for targeted therapies.

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