Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding
Copyright policy )Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding
The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell.
- Johns Hopkins Medicine United States
- University of Wisconsin–Oshkosh United States
- Johns Hopkins University United States
- University of Wisconsin–Madison United States
- Johns Hopkins University School of Medicine United States
Models, Molecular, Magnetic Resonance Spectroscopy, ATPase; Copper; Membrane Protein;, Blotting, Western, Molecular Sequence Data, Animals, Humans, Amino Acid Sequence, Cation Transport Proteins, Protein Dynamic, Adenosine Triphosphatases, Binding Sites, Microscopy, Confocal, Cell Membrane, Protein Structure, Tertiary, Luminescent Proteins, Protein Transport, HEK293 Cells, Membrane Trafficking, Copper-Transporting ATPases, Nanobody; Nuclear Magnetic Resonance (NMR);, Camelids, New World, Copper, Protein Binding
Models, Molecular, Magnetic Resonance Spectroscopy, ATPase; Copper; Membrane Protein;, Blotting, Western, Molecular Sequence Data, Animals, Humans, Amino Acid Sequence, Cation Transport Proteins, Protein Dynamic, Adenosine Triphosphatases, Binding Sites, Microscopy, Confocal, Cell Membrane, Protein Structure, Tertiary, Luminescent Proteins, Protein Transport, HEK293 Cells, Membrane Trafficking, Copper-Transporting ATPases, Nanobody; Nuclear Magnetic Resonance (NMR);, Camelids, New World, Copper, Protein Binding
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