Interactions Between Metal-Binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding [Molecular Biophysics]

September 24th, 2014 by Huang, Y., Nokhrin, S., Hassanzadeh-Ghassabeh, G., Yu, C. H., Yang, H., Barry, A. N., Tonelli, M., Markley, J. L., Muyldermans, S., Dmitriev, O. Y., Lutsenko, S.

The biologically and clinically important membrane transporters are challenging proteins to study owing to their low level of expression, multi-domain 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 the metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B towards 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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Interactions Between Metal-Binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding [Molecular Biophysics]