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Aptameric Probe Specifically Binding Protein Heterodimer Rather Than Monomers.

ABSTRACT: Dimerization of proteins occurs frequently and plays integral roles in biological processes. However, no single molecular probe is available for in situ detection of protein dimers on cells and tissues because of the difficulty of isolating complete protein dimers for probe preparation and screening, which has greatly hampered the biomedical study of protein dimers. Herein, a G-rich DNA aptamer (termed BG2) that only binds alkaline phosphatase (AP) heterodimers rather than monomers is reported. This aptamer is generated by the cell-SELEX (systematic evolution of ligands by exponential enrichment) technique and proves to fold into a duplex stabilized antiparallel G-quadruplex structure. Using BG2 as molecular probe, AP heterodimers are found to be expressed on several kinds of cancer cells. As an affinity ligand, BG2 could isolate AP heterodimers from cell lysate. BG2 is also demonstrated to be applicable for tumor imaging in mice xenografted with cells highly expressing AP heterodimers. AP isozymes are found in several tissues and blood throughout the body, but the function and tissue distribution of AP heterodimers are totally unknown; therefore, BG2 could serve as a molecular probe to uncover the mystery of AP heterodimers. The generation of aptameric probes by cell-SELEX will open up a new situation for the study of protein dimers.

SUBMITTER: Bing T

PROVIDER: S-EPMC6548965 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Aptameric Probe Specifically Binding Protein Heterodimer Rather Than Monomers.

Bing Tao T Shen Luyao L Wang Junyan J Wang Linlin L Liu Xiangjun X Zhang Nan N Xiao Xiao X Shangguan Dihua D

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20190409 11

Dimerization of proteins occurs frequently and plays integral roles in biological processes. However, no single molecular probe is available for in situ detection of protein dimers on cells and tissues because of the difficulty of isolating complete protein dimers for probe preparation and screening, which has greatly hampered the biomedical study of protein dimers. Herein, a G-rich DNA aptamer (termed BG2) that only binds alkaline phosphatase (AP) heterodimers rather than monomers is reported. ...[more]

PMID: 31179220

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Project description:We have presented a new protein-protein docking approach to model heterodimeric structures based on the conformations of the monomeric units. The conventional modeling method relies on superimposing two monomeric structures onto the crystal structure of a homologous protein dimer. The resulting structure may exhibit severe backbone clashes at the dimeric interface depending on the backbone dissimilarity between the target and template proteins. Our method overcomes the backbone clashing problem and requires no a priori knowledge of the dimeric structure of a homologous protein. Here we used human Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel whose dysfunction causes cystic fibrosis, for illustration. The two intracellular nucleotide-binding domains (NBDs) of CFTR control the opening and closing of the channel. Yet, the structure of the CFTR's NBD1-NBD2 complex has not been experimentally determined. Thus, correct modeling of this heterodimeric structure is valuable for understanding CFTR functions and would have potential applications for drug design for cystic fibrosis treatment. Based on the crystal structure of human CFTR's NBD1, we constructed a model of the NBD1-NBD2 complex. The constructed model is consistent with the dimeric mode observed in the crystal structures of other ABC transporters. To verify our structural model, an ATP substrate was docked into the nucleotide-binding site. The predicted binding mode shows consistency with related crystallographic findings and CFTR functional studies. Finally, genistein, an agent that enhances CFTR activity, though the mechanism for such enhancement is unclear, was docked to the model. Our predictions agreed with genistein's bell-shaped dose-response relationship. Potential mutagenesis experiments were proposed for understanding the potentiation mechanism of genistein and for providing insightful information for drug design targeting at CFTR. The method used in this study can be applied to modeling studies of other dimeric protein structures.

Dataset Information

Aptameric Probe Specifically Binding Protein Heterodimer Rather Than Monomers.

Publications

Aptameric Probe Specifically Binding Protein Heterodimer Rather Than Monomers.

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