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C-terminal Tail (c-terminal + tail)
Selected AbstractsStructure and function of AMP-activated protein kinaseACTA PHYSIOLOGICA, Issue 1 2009J. S. Oakhill Abstract AMP-activated protein kinase (AMPK) regulates metabolism in response to energy demand and supply. AMPK is activated in response to rises in intracellular AMP or calcium-mediated signalling and is responsible for phosphorylating a wide variety of substrates. Recent structural studies have revealed the architecture of the ,,, subunit interactions as well as the AMP binding pockets on the , subunit. The , catalytic domain (1,280) is autoinhibited by a C-terminal tail (313,335), which is proposed to interact with the small lobe of the catalytic domain by homology modelling with the MARK2 protein structure. Two direct activating drugs have been reported for AMPK, the thienopyridone compound A769662 and PTI, which may activate by distinct mechanisms. [source] The Arabidopsis class VIII myosin ATM2 is involved in endocytosisCYTOSKELETON, Issue 6 2008Amirali Sattarzadeh Abstract Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens -based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] DAP kinase activity is critical for C2 -ceramide-induced apoptosis in PC12 cellsFEBS JOURNAL, Issue 1 2002Mutsuya Yamamoto Exposure of PC12 cells to C2 -ceramide results in dose-dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-,-induced apoptosis of HeLa cells, in the C2 -ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 µm C2 -ceramide, both the total (assayed in the presence of Ca2+/calmodulin) and Ca2+/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 µm C2 -ceramide, the Ca2+/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C2 -ceramide concentration. C8 -ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C2 -dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C2 - and C8 -ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C2 -, and C8 -ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis. [source] Nuclear import and DNA-binding activity of RFX1FEBS JOURNAL, Issue 10 2001Evidence for an autoinhibitory mechanism RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent ,acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC. [source] The carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neuronesGENES TO CELLS, Issue 2 2005Qiuping Hu A recent report on the mechanism of synaptic targeting of Cav2.2 channel suggested that this process depends upon the presence of long C-terminal tail and that protein interactions mediated by SH3-binding and PDZ-binding motifs in the tail region are important. To examine the possibility that C-terminal tail of the Cav2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Cav2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Cav2.1, and HS and HL encode Cav2.1 channel with a long C-terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ-binding motif located at the C-terminus of the long version of Cav2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C-terminal region is not indispensable for the subcellular localization of Cav2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Cav2.1 channel is different from that of Cav2.2, though these channels share various structural and functional characteristics. [source] Familial Hypocalciuric Hypercalcemia Caused by an R648stop Mutation in the Calcium-Sensing Receptor Gene ,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2002Mika Yamauchi Abstract In this study, we report an 84-year-old female proband in a Japanese family with familial hypocalciuric hypercalcemia (FHH) caused by an R648stop mutation in the extracellular calcium-sensing receptor (CaR) gene. At the age of 71 years, she presented with hypercalcemia (11.4 mg/dl), hypocalciuria (Cca/Ccr = 0.003), hypermagnesemia (2.9 mg/dl), and a high-serum parathyroid hormone (PTH) level (midregion PTH, 3225 [160,520] pg/ml). At the age of 74 years, a family screening was carried out and revealed a total of 9 hypercalcemic individuals (all intact PTH values <62 pg/dl) among 17 family members tested, thus, being diagnosed as FHH. Two and one-half of three clearly enlarged parathyroid glands were resected, because persistently high PTH levels (intact PTH, 292 pg/ml; midregion PTH, 5225 pg/ml) and the presence of a markedly enlarged parathyroid gland by several imaging modalities (ultrasonography, computed tomography [CT], magnetic resonance imaging [MRI], and subtraction scintigraphy) suggested coexistent primary hyperparathyroidism (pHPT); however, hypercalcemia persisted postoperatively. Histological and immunohistochemical examination revealed that the resected parathyroid glands showed lipohyperplasia as well as normally expressed Ki67, vitamin D receptor (VDR), and the CaR. Sequence analysis disclosed that the proband and all affected family members had a heterozygous nonsense (R648stop) mutation in the CaR gene. This mutation is located in the first intracellular loop; thus, it would be predicted to produce a truncated CaR having only one transmembrane domain (TMD) and lacking its remaining TMDs, intracellular loops, and C-terminal tail. Western analysis of biotinylated HEK293 cells transiently transfected with this mutant receptor showed cell surface expression of the truncated protein at a level comparable with that of the wild-type CaR. The mutant receptor, however, exhibited no increase in intracellular free calcium concentration (Ca2+i) when exposed to high extracellular calcium concentrations (Ca2+o). The proband's clinical course was complicated because of associated renal tubular acidosis (RTA) and nephrotic syndrome. However, it was unclear whether their association affected the development of elevated serum PTH and parathyroid gland enlargement. This report is the first to show that an R648stop CaR mutation yields a truncated receptor that is expressed on the cell surface but is devoid of biological activity, resulting in FHH. [source] Role of Src in ligand-specific regulation of ,-opioid receptor desensitization and internalizationJOURNAL OF NEUROCHEMISTRY, Issue 1 2009Min-Hua Hong Abstract The opioid receptors are a member of G protein-coupled receptors that mediate physiological effects of endogenous opioid peptides and structurally distinct opioid alkaloids. Although it is well characterized that there is differential receptor desensitization and internalization properties following activation by distinct agonists, the underlying mechanisms remain elusive. We investigated the signaling events of ,-opioid receptor (,OR) initiated by two ligands, DPDPE and TIPP. We found that although both ligands inhibited adenylyl cyclase (AC) and activated ERK1/2, only DPDPE induced desensitization and internalization of the ,OR. We further found that DPDPE, instead of TIPP, could activate GRK2 by phosphorylating the non-receptor tyrosine kinase Src and translocating it to membrane receptors. Activation of GRK2 led to the phosphorylation of serine residues in the C-terminal tail, which facilitates ,-arrestin1/2 membrane translocation. Meanwhile, we also found that DPDPE promoted ,-arrestin1 dephosphorylation in a Src-dependent manner. Thus, DPDPE appears to strengthen ,-arrestin function by dual regulations: promoting ,-arrestin recruitment and increasing ,-arrestin dephosphorylation at the plasma membrane in a Src-dependent manner. All effects initiated by DPDPE could be abolished or suppressed by PP2, an inhibitor of Src. Morphine, which has been previously shown to be unable to desensitize or internalize ,OR, also behaved as TIPP in failure to utilize Src to regulate ,OR signaling. These findings point to the existence of agonist-specific utilization of Src to regulate ,OR signaling and reveal the molecular events by which Src modulates ,OR responsiveness. [source] C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processesJOURNAL OF PINEAL RESEARCH, Issue 2 2008Shalini Sethi Abstract:, Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G-protein-coupled melatonin receptors, MT1 and MT2. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site-directed mutagenesis. Two mutations were constructed in the cytoplasmic C-terminal tail of each receptor subtype: (i) a cysteine residue in the C-terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT1C7.72A and MT2C7.77A) and (ii) the C-terminal tail in the MT1 and MT2 receptors was truncated, removing the putative phosphorylation and ,-arrestin binding sites (MT1Y7.64 and MT2Y7.64). These mutations did not alter the affinity of 2-[125I]-iodomelatonin binding to the MT1 or MT2 receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3,,5,-cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C-terminal tail of both receptors (MT1Y7.64 and MT2Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions. [source] A degradation signal recognition in prokaryotesJOURNAL OF SYNCHROTRON RADIATION, Issue 3 2008Eun Young Park The degradation of ssrA-tagged substrates in prokaryotes is conducted by a subset of ATP-dependent proteases, including ClpXP complex. More than 630 sequences of ssrA have been identified from 514 species, and are conserved in a wide range of prokaryotes. SspB protein markedly stimulates the degradation of these ssrA-tagged substrates by the ClpXP proteolytic machine. The dimeric SspB protein is composed of a compact ssrA-binding domain, which has a dimerization surface and a flexible C-terminal tail with a ClpX-binding motif at its very end. Since SspB is an adaptor protein for the ClpXP complex, designed mutagenesis, fluorescence spectroscopy, biochemistry and X-ray crystallography have been used to investigate the mechanism of delivery of ssrA-tagged proteins. In this paper the structural basis of ssrA-tag recognition by ClpX and SspB, as well as SspB-tail recognition by ZBD, is described. [source] Molecular mechanisms of cross-inhibition between nicotinic acetylcholine receptors and P2X receptors in myenteric neurons and HEK-293 cellsNEUROGASTROENTEROLOGY & MOTILITY, Issue 8 2010D. A. Decker Abstract Background, P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and ,3,4subunit-containing nAChRs. Methods, The function of P2X2 and ,3,4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Key Results, In HEK-293 cells expressing ,3,4 nAChRs and P2X2 receptors, co-application of ATP and acetylcholine caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing ,3,4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR) (P > 0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing ,3,4 nAChRs transfected with P2X2TR. Currents caused by low (,30 ,mol L,1), but not high (,100 ,mol L,1) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with ,3,4 nAChRs. Conclusions & Inferences, The C-terminal tail of P2X2 receptors mediates cross-inhibition between ,3,4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross-inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. [source] Synthesis and Spectroscopic Characterization of Photo-affinity Peptide Ligands to Study Rhodopsin,G Protein Interaction,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Yihui Chen G protein-coupled receptors (GPCRs) are involved in the control of virtually all aspects of our behavior and physiology. Activated receptors catalyze nucleotide exchange in heterotrimeric G proteins (composed of ,·GDP, , and , subunits) on the inner surface of the cell membrane. The GPCR rhodopsin and the G protein transducin (Gt) are key proteins in the early steps of the visual cascade. The main receptor interaction sites on Gt are the C-terminal tail of the Gt,-subunit and the farnesylated C-terminal tail of the Gt,-subunit. Synthetic peptides derived from these C-termini specifically bind and stabilize the active rhodopsin conformation (R*). Here we report the synthesis of R*-interacting peptides containing photo-reactive groups with a specific isotope pattern, which can facilitate detection of cross-linked products by mass spectrometry. In a preliminary set of experiments, we characterized such peptides derived from the farnesylated Gt, C-terminus (Gt,(60-71)far) in terms of their capability to bind R*. Here, we describe novel peptides with photo-affinity labels that bind R* with affinities similar to that of the native Gt,(60-71)far peptide. Such peptides will enable an improved experimental strategy to probe rhodopsin,Gt interaction and to map so far unknown interaction sites between both proteins. [source] An electrostatic network and long-range regulation of Src kinasesPROTEIN SCIENCE, Issue 11 2008Elif Ozkirimli Abstract The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain,domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains. [source] The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain bindingPROTEIN SCIENCE, Issue 4 2007Shugui Chen Abstract The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis -interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain. [source] Secondary structure and dynamics of micelle bound ,- and ,-synucleinPROTEIN SCIENCE, Issue 5 2006Yoon-hui Sung Abstract We have used solution state NMR spectroscopy to characterize the secondary structure and backbone dynamics of the proteins ,- and ,-synuclein in their detergent micelle-bound conformations. Comparison of the results with those previously obtained for the Parkinson's disease-linked protein ,-synuclein shows that structural differences between the three homologous synuclein family members are directly related to variations in their primary amino acid sequences. An 11-residue deletion in the lipid-binding domain of ,-synuclein leads to the destabilization of an entire segment of the micelle-bound helical structure containing the deletion site. The acidic C-terminal tail region of ,-synuclein, which displays extensive sequence divergence, is more highly disordered than the corresponding regions in the other two family members. The observed structural differences are likely to mediate functional variations between the three proteins, with differences between ,- and ,-synuclein expected to revolve around their lipid interactions, while differences in ,-synuclein function are expected to result from different protein,protein interactions mediated by its unique C-terminal tail. [source] Homology modeling and molecular dynamics simulations of lymphotactinPROTEIN SCIENCE, Issue 11 2000Buyong Ma Abstract We have modeled the structure of human lymphotactin (hLpnt), by homology modeling and molecular dynamics simulations. This chemokine is unique in having a single disulfide bond and a long C-terminal tail. Because other structural classes of chemokines have two pairs of Cys residues, compared to one in Lpnt, and because it has been shown that both disulfide bonds are required for stability and function, the question arises how the Lpnt maintains its structural integrity. The initial structure of hLpnt was constructed by homology modeling. The first 63 residues in the monomer of hLpnt were modeled using the structure of the human CC chemokine, RANTES, whose sequence appeared most similar. The structure of the long C-terminal tail, missing in RANTES, was taken from the human muscle fatty-acid binding protein. In a Protein Data Bank search, this protein was found to contain a sequence that was most homologous to the long tail. Consequently, the modeled hLpnt C-terminal tail consisted of both ,-helical and ,-motifs. The complete model of the hLpnt monomer consisted of two ,-helices located above the five-stranded ,-sheet. Molecular dynamics simulations of the solvated initial model have indicated that the stability of the predicted fold is related to the geometry of Pro78. The five-stranded ,-sheet appeared to be preserved only when Pro78 was modeled in the cis conformation. Simulations were also performed both for the C-terminal truncated forms of the hLpnt that contained one or two (CC chemokine-like) disulfide bonds, and for the chicken Lpnt (cLpnt). Our MD simulations indicated that the turn region (T30-G34) in hLpnt is important for the interactions with the receptor, and that the long C-terminal region stabilizes both the turn (T30-G34) and the five-stranded ,-sheet. The major conclusion from our theoretical studies is that the lack of one disulfide bond and the extension of the C-terminus in hLptn are mutually complementary. It is very likely that removal of two Cys residues sufficiently destabilizes the structure of a chemokine molecule, particularly the core ,-sheet, to abolish its biological function. However, this situation is rectified by the long C-terminal segment. The role of this long region is most likely to stabilize the first ,-turn region and ,-helix H1, explaining how this chemokine can function with a single disulfide bond. [source] Modulation of Ca2+ signalling in rat atrial myocytes: possible role of the ,1c carboxyl terminalTHE JOURNAL OF PHYSIOLOGY, Issue 2 2003Sun-Hee Woo Ca2+ influx through L-type Cav1.2 (,1c) Ca2+ channels is a critical step in the activation of cardiac ryanodine receptors (RyRs) and release of Ca2+ via Ca2+ -induced Ca2+ release(CICR). The released Ca2+, in turn, is the dominant determinant of inactivation of the Ca2+ current (ICa) and termination of release. Although Ca2+ cross-signalling is mediated by high Ca2+ fluxes in the microdomains of ,1c -RyR complexes, ICa -gated Ca2+ cross-signalling is surprisingly resistant to intracellular Ca2+ buffering and has steeply voltage-dependent gain, inconsistent with a strict CICR mechanism, suggesting the existence of additional regulatory step(s). To explore the possible regulatory role of the carboxyl (C)-terminal tail of ,1c in modulating Ca2+ signalling, we tested the effects of introducing two ,1c C-terminal peptides, LA (1571,1599) and K (1617,1636) on the central ,1c -unassociated Ca2+ -release sites of atrial myocytes, using rapid (240 Hz) two-dimensional confocal Ca2+ imaging. The frequency of spontaneously activating central sparks increased by approximately fourfold on dialysing LA- but not K-peptide into myocytes voltage-clamped at -80 mV. The rate but not the magnitude of caffeine (10 mM)-triggered central Ca2+ release was significantly accelerated by LA- but not K-peptide. Individual Ca2+ spark size and flux were larger in LA- but not in K-peptide-dialysed myocytes. Although LA-peptide did not change the amplitude or inactivation kinetics of ICa, LA-peptide did strongly enhance the central Ca2+ transients triggered by ICa at -30 mV (small ICa) but not at +20 mV (large ICa). In contrast, K-peptide had no effect on either ICa or the local Ca2+ transients. LA-peptide with a deleted calmodulin-binding region (LM1-peptide) had no significant effects on the central spark frequency but suppressed spontaneous spark frequency in the periphery. Our results indicate that the calmodulin-binding LA motif of the ,1c C-terminal tail may sensitize the RyRs, thereby increasing their open probability and providing for both the voltage-dependence of CICR and the higher frequency of spark occurrence in the periphery of atrial myocytes where the native ,1c -RyR complexes are intact. [source] Structures of the PKC-, kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533,551 in the C-terminal tail and their roles in ATP bindingACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Tetsuo Takimura Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-,, PKC-, and PKC-,II kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC,substrate complex has been determined. In the previously determined PKC-, complex, residues 533,551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-, kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0,Å resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-, are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-,, which is different from the case of the prototypical AGC-branch kinase PKA. [source] Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2005Sheila Gover Human glucose-6-phosphate dehydrogenase (G6PD) is NADP+ -dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, ,G6PD, with glucose-6-phosphate and NADP+ have been crystallized and their structures solved to 2.9 and 2.5,Å, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PDCanton) in which NADP+ is bound at the structural site. Substrate binding in ,G6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP+ binding at the coenzyme site is seen to be comparable to NADP+ binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer,dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP+ binds in a very similar way in the ,G6PD,NADP+ complex and in G6PDCanton, while in the substrate complex the structural NADP+ has low occupancy and the C-terminal tail at the structural NADP+ site is disordered. The implications of possible interaction between the structural NADP+ and G6P are considered. [source] Characterization of sequence variations in human histone H1.2 and H1.4 subtypesFEBS JOURNAL, Issue 14 2005Bettina Sarg In humans, eight types of histone H1 exist (H1.1,H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC,GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA,AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC,GTC shift, indicating that this is a relatively frequent polymorphism. The AAA,AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions. [source] Heterodimerization of opioid receptor-like 1 and µ-opioid receptors impairs the potency of µ receptor agonistJOURNAL OF NEUROCHEMISTRY, Issue 6 2005Hung-Li Wang Abstract Nociceptin activation of ORL1 (opioid receptor-like 1 receptor) has been shown to antagonize µ receptor-mediated analgesia at the supraspinal level. ORL1 and µ-opioid receptor (µR) are co-expressed in several subpopulations of CNS neurons involved in regulating pain transmission. The amino acid sequence of ORL1 also shares a high degree of homology with that of µ receptor. Thus, it is hypothesized that ORL1 and µR interact to form the heterodimer and that ORL1/µR heterodimerization may be one molecular basis for ORL1-mediated antiopioid effects in the brain. To test this hypothesis, myc-tagged ORL1 and HA-tagged µR are co-expressed in human embryonic kidney (HEK) 293 cells. Co-immunoprecipitation experiments demonstrate that ORL1 dimerizes with µR and that intracellular C-terminal tails of ORL1 and µR are required for the formation of ORL1/µR heterodimer. Second messenger assays further indicate that formation of ORL1/µR heterodimer selectively induces cross-desensitization of µR and impairs the potency by which [d -Ala2,N -methyl-Phe4,Gly-ol5]enkephalin (DAMGO) inhibits adenylate cyclase and stimulates p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. These results provide the evidence that ORL1/µR heterodimerization and the resulting impairment of µ receptor-activated signaling pathways may contribute to ORL1-mediated antiopioid effects in the brain. [source] The 1.30,Å resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimerACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000Jane E. Ladner The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30,Å resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P212121 with unit-cell parameters a = 52.2, b = 83.8, c = 86.0,Å. The ABC trimer of the monoclinic crystal structure [Chook et al. (1994), J. Mol. Biol.240, 476,500] was used as the starting model. The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues. In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure. This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an Rfree of 0.236. The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site. This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection. A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog. In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice. [source] | ||||||||||||||||