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Wu DY  Yao Z 《Cell research》2006,16(3):319-322
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Two non-stoichiometric binding sites had previously been characterized for the NK-1 receptor using two different types of radiolabelled analogues of substance P. However, the question remained on their eventual conformational interconversion induced or not by the ligand. In this study, kinetic, saturation, and competition studies using [3H]propionyl[Pro(9)]SP demonstrate the existence of two independent binding components in CHO cells transfected with the human NK-1 receptor, with K(d) values of 0.040 nM ( approximately 20% of total sites) and 5.9 nM ( approximately 80% of total sites) that correspond to those of the two previously described binding sites. These two binding sites do not seem to interconvert since the minor one can be selectively extinguished in saturation studies in the presence of a SP analogue specific of this binding site.  相似文献   

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Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.  相似文献   

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Negron C  Fufezan C  Koder RL 《Proteins》2009,74(2):400-416
Helical bundles which bind heme and porphyrin cofactors have been popular targets for cofactor-containing de novo protein design. By analyzing a highly nonredundant subset of the protein databank we have determined a rotamer distribution for helical histidines bound to heme cofactors. Analysis of the entire nonredundant database for helical sequence preferences near the ligand histidine demonstrated little preference for amino acid side chain identity, size, or charge. Analysis of the database subdivided by ligand histidine rotamer, however, reveals strong preferences in each case, and computational modeling illuminates the structural basis for some of these findings. The majority of the rotamer distribution matches that predicted by molecular simulation of a single porphyrin-bound histidine residue placed in the center of an all-alanine helix, and the deviations explain two prominent features of natural heme protein binding sites: heme distortion in the case of the cytochromes C in the m166 histidine rotamer, and a highly prevalent glycine residue in the t73 histidine rotamer. These preferences permit derivation of helical consensus sequence templates which predict optimal side chain-cofactor packing interactions for each rotamer. These findings thus promise to guide future design endeavors not only in the creation of higher affinity heme and porphyrin binding sites, but also in the direction of bound cofactor geometry.  相似文献   

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The chaperone-related p97 protein plays a central role in various cellular processes involving the ubiquitin-proteasome system. The diverse functions of p97 are controlled by a large number of cofactors that recruit specific substrates or influence their ubiquitylation state. Many cofactors bind through a UBX or PUB domain, two major p97 binding modules. However, the recently identified UBXD1 cofactor possesses both domains. To elucidate the molecular basis underlying the interaction between UBXD1 and p97, we analyzed the contribution of both domains to p97 binding biochemically and in living cells. The PUB domain mediated robust binding to the carboxy-terminus of p97, while the UBX domain did not contribute to p97 binding. Importantly, we identified an additional p97 binding site in UBXD1 that competed with the p47 cofactor for binding to the N domain of p97. This novel, bipartite binding mode suggests that UBXD1 could be an efficient regulator of p97 cofactor interactions.  相似文献   

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An Sp1/Sp3 binding polymorphism confers methylation protection   总被引:1,自引:0,他引:1  
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