Nanobodies against the metal binding domains of ATP7B as tools to study copper transport in the cell
文献信息
Eva-Maria E. Uhlemann, Corey H. Yu, Jaala Patry, Natalia Dolgova, Svetlana Lutsenko, Serge Muyldermans, Oleg Y. Dmitriev
Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10−9 to 10−6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.
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Metallomics
Metallomics publishes cutting-edge investigations aimed at elucidating the identification, distribution, dynamics, role and impact of metals and metalloids in biological systems. Studies that address the “what, where, when, how and why” of these inorganic elements in cells, tissues, organisms, and various environmental niches are welcome, especially those employing multidisciplinary approaches drawn from the analytical, bioinorganic, medicinal, environmental, biophysical, cell biology, plant biology and chemical biology communities. We are particularly interested in articles that enhance our chemical and/or physical understanding of the molecular mechanisms of metal-dependent life processes, and those that probe the common space between metallomics and other ‘omics approaches to uncover new insights into biological processes. Metallomics seeks to position itself at the forefront of those advances in analytical chemistry destined to clarify the enormous complexity of biological systems. As such, we particularly welcome those papers that outline cutting-edge analytical technologies, e.g., in the development and application of powerful new imaging, spectroscopic and mass spectrometric modalities. Work that describes new insights into metal speciation, trafficking and dynamics in complex systems or as a function of microenvironment are also strongly encouraged. Studies that examine the interconnectivity of metal-dependent processes with systems level responses relevant to organismal health or disease are also strongly encouraged, for example those that probe the effect of chemical exposure on metal homeostasis or the impact of metal-based drugs on cellular processes.
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