title: Interactions between metal-binding domains modulate intracellular targeting of Cu(I)-ATPase ATP7B, as revealed by nanobody binding.
abstract: 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.
authors.affiliation: From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Huang
authors.affiliation: the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Sergiy
authors.initials: S
authors.lastname: Nokhrin
authors.affiliation: the Vrije Universiteit Brussel, Structural Biology Research Center, and Nanobody Service Facility, VIB, 1050 Brussels, Belgium, and.
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Hassanzadeh-Ghassabeh
authors.affiliation: the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Corey H
authors.initials: CH
authors.lastname: Yu
authors.affiliation: From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Haojun
authors.initials: H
authors.lastname: Yang
authors.affiliation: From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Amanda N
authors.initials: AN
authors.lastname: Barry
authors.affiliation: the Department of Biochemistry, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Marco
authors.initials: M
authors.lastname: Tonelli
authors.affiliation: the Department of Biochemistry, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
authors.affiliation: the Vrije Universiteit Brussel, Structural Biology Research Center, and.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada, Oleg.Dmitriev@usask.ca.
authors.forename: Oleg Y
authors.initials: OY
authors.lastname: Dmitriev
authors.affiliation: From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, Lutsenko@jhmi.edu.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Lutsenko
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002161:Camelids, New World; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D008164:Luminescent Proteins; D061905:Single-Domain Antibodies; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases
keywords: ATPase; Copper; Membrane Protein; Membrane Trafficking; Nanobody; Nuclear Magnetic Resonance (NMR); Protein Dynamic