Synthetic Domain Peptide Research Articles

Direct Detection of Domain Peptide Binding to the Cardiac Ryanodine Receptor (RYR2) using FRET

The RyR2 Ca release channel is activated by the synthetic domain peptide DPc10, which corresponds to a 36-residue sequence within the channel's central mutation hot-spot region. DPc10 activation is hypothesized to result from the destabilization of a critical intramolecular interaction between the N-terminal and central hot-spot regions of the full-length RyR2, and to thereby mimic the effects of arrhythmogenic mutations on this putative intramolecular interaction controlling channel activation. However, the site of DPc10 binding within the RyR2 3D structure is uncertain, and factors that may modulate binding are undefined. To directly monitor and map DPc10 binding to the RyR2, we attached a FRET acceptor at DPc10's N-terminus. A FRET donor was targeted to the RyR2 cytoplasmic assembly via a fluorescent-labeled FKBP12.6. Addition of the acceptor-labeled DPc10 (30 µM) resulted in a marked decrease in fluorescence of the RyR2-bound FKBP12.6. Fluorescence was partially restored upon FKBP12.6 dissociation from the RyR2, indicating that a major fraction of the total fluorescence decrease was attributable to FRET between FKBP12.6 and DPc10 when bound to the channel. The DPc10 dependence of FRET was similar to the DPc10 dependence of RyR2 activation observed previously in bilayer and ryanodine binding studies (EC50 ∼25 µM), consistent with the likelihood that FRET reflected DPc10 binding at its regulatory site on the RyR2. FRET decreased as a function of increasing Ca (30 nM to 300 µM), suggesting that Ca activation of the RyR2 altered either the affinity of DPc10 binding or its proximity to the FKBP12.6 subunit. We conclude that DPc10 binds to a site on the RyR2 within 10 nm of FKBP12.6. Regulatory interactions and structural changes at this site can be monitored using FRET.

Abstract 576: Defective Domain-Domain Interaction Between C-Terminal and Central Regions of Ryanodine Receptor as a Critical Cause of Diastolic Ca 2+ Spark and Delayed Afterdepolarization

Ryanodine receptor-2 (RyR2) central domain peptide, DPc10 (Gly 2460 -Pro 2495 ), was found to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between N-terminal 1– 600 and central domains 2000 –2500 . Using the domain peptide approach, here, we investigated how single point mutation at C-terminal region in CPVT influences on intracellular Ca 2+ handling. Methods and Results: Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (n= 5), then fluorescently labeled with methylcoumarin acetate (MCA) using DP 4090 – 4123 , a synthetic domain peptide corresponding to Pro-Glu (mutable domain in CPVT), as a site-directing carrier; the carrier was removed after MCA labeling. The DP 4090 – 4123 mediated a specific MCA fluorescence labeling of RyR2. After tryptic digestion, the major MCA-fluorescently labeled fragment of RyR2 (150 kD) was detected by an antibody (Ab) against central region (Ab 2163 ), but not by Ab 2808, N-terminal (Ab 10 ) and C-terminal region (Ab 5030 ). Addition of DP 4090 – 4123 to the MCA-labeled SR induced domain unzipping between C-terminal and its putative counter domains (EC50=30μM), as confirmed by a marked quenching of the MCA fluorescence by a large-size fluorescence quencher. The DP 4090 – 4123 induced a prominent SR Ca 2+ leak (EC50=30μM). In DP 4090 – 4123 -incoporated cardiomyocytes (by protein delivery kit), diastolic Ca 2+ spark frequency was markedly increased (4.0±1.0 s -1 100ìm -1 , p<0.01), compared to normal cardiomyocytes (1.4±0.4 s -1 100ìm -1 ), and after addition of 100nM forskolin both Ca2+ wave and delayed afterdepolarization-mediated Ca 2+ transient (DAD-CaT) were also frequently observed (but not in normal cardiomyocytes). All of these effects of DP 4090 – 4123 were abolished either by addition of 10 μM tetracaine or by Asn-to-Lys mutation made in DP 4090 – 4123 , mimicking human CPVT mutation. Conclusions: Domain-domain interaction between C-terminal and central domains (within 1200 –2850) seems to play an important role in channel gating of RyR2, and its defectiveness by point mutation may induce diastolic Ca 2+ spark and delayed afterdepolarization that triggers lethal arrhythmia.

Probing a putative dantrolene-binding site on the cardiac ryanodine receptor

Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [3H]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photolabelled by [3H]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [3H]dantrolene to cardiac SR as a function of free Ca2+. We found that > or =10 mM EGTA increased [3H]dantrolene binding to RyR2 by approximately 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor

Dantrolene is a drug that suppresses intracellular Ca(2+) release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca(2+) release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [(3)H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [(3)H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro and in vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [(3)H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1-2s in both Western blots and immunoprecipitation assays and specifically inhibits [(3)H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590-609.

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.

Postulated Role of Interdomain Interaction within the Ryanodine Receptor in Ca2+ Channel Regulation

Localized distribution of malignant hyperthermia (MH) and central core disease (CCD) mutations in N-terminal and central domains of the ryanodine receptor suggests that the interaction between these domains may be involved in Ca(2+) channel regulation. To test this hypothesis, we investigated the effects of a new synthetic domain peptide DP4 corresponding to the Leu(2442)-Pro(2477) region of the central domain. DP4 enhanced ryanodine binding and induced a rapid Ca(2+) release. The concentration for half-maximal activation by agonists was considerably reduced in the presence of DP4. These effects of DP4 are analogous to the functional modifications of the ryanodine receptor caused by MH/CCD mutations (viz. hyperactivation of the channel and hypersensitization of the channel to agonists). Replacement of Arg of DP4 with Cys, mimicking the in vivo Arg(2458)-to-Cys(2458) mutation, abolished the activating effects of DP4. An N-terminal domain peptide DP1 (El-Hayek, R., Saiki, Y., Yamamoto, T., and Ikemoto, N. (1999) J. Biol. Chem. 274, 33341-33347) shows similar activation/sensitization effects. The addition of both DP4 and DP1 produced mutual interference of their activating functions. We tentatively propose that contact between the two (N-terminal and central) domains closes the channel, whereas removal of the contact by these domain peptides or by MH/CCD mutations de-blocks the channel, resulting in hyperactivation/hyper-sensitization effects.

Secretion of functional salivary peptide by Streptococcus gordonii which inhibits fimbria-mediated adhesion of Porphyromonas gingivalis.

Porphyromonas gingivalis, a putative periodontopathogen, can bind to human salivary components with its fimbriae. We have previously shown that fimbriae specifically bind to a peptide domain shared by a major salivary component, i.e., proline-rich (glyco)proteins (PRPs). The synthetic domain peptide PRP-C (pPRP-C) significantly inhibits the fimbrial binding to PRPs. In this study, a recombinant strain of Streptococcus gordonii secreting pPRP-C was generated as a model of a possible approach to prevent the oral colonization by the pathogen. A duplicate DNA fragment (prpC) encoding pPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides. prpC was connected downstream to a promoter and a gene encoding a signal peptide of Streptococcus downei glucosyltransferase I in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was inserted to produce the transformant S. gordonii G9B, which then successfully secreted recombinant pPRP-C (r-pPRP-C) of the expected size. The concentrated bacterial culture supernatant containing r-pPRP-C inhibited the binding of P. gingivalis cells and fimbriae to PRP1 in a dose-dependent manner up to 72 and 77%, respectively. The r-pPRP-C concentrate also inhibited the coaggregation of P. gingivalis with various streptococcal strains as effectively as synthetic pPRP-C in a dose-dependent manner. Collectively, pPRP-C was found to be able to prevent P. gingivalis adherence to salivary receptor protein and plaque-forming bacteria. These results suggest that this recombination approach with a nonperiodontopathic bacterium may be suitable for the therapeutic prevention of P. gingivalis adherence to the oral cavity.