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T versus D in the MTCXXC motif of copper transport proteins plays a role in directional metal transport
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2014 (English)In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 6, 1037-1047 p.Article in journal (Refereed) Published
Abstract [en]

To avoid toxicity and control levels of metal ions, organisms have developed specific metal transport systems. In humans, the cytoplasmic Cu chaperone Atox1 delivers Cu to metal-binding domains of ATP7A/B in the Golgi, for incorporation into Cu-dependent proteins. The Cu-binding motif in Atox1, as well as in target Cu-binding domains of ATP7A/B, consists of a MX1CXXC motif where X-1 = T. The same motif, with X-1 = D, is found in metal-binding domains of bacterial zinc transporters, such as ZntA. The Asp is proposed to stabilize divalent over monovalent metals in the binding site, although metal selectivity in vivo appears predominantly governed by protein-protein interactions. To probe the role of T versus D at the X-1 position for Cu transfer in vitro, we created MDCXXC variants of Atox1 and the fourth metal-binding domain of ATP7B, WD4. We find that the mutants bind Cu like the wild-type proteins, but when mixed, in contrast to the wild-type pair, the mutant pair favors Cu-dependent hetero-dimers over directional Cu transport from Atox1 to WD4. Notably, both wild-type and mutant proteins can bind Zn in the absence of competing reducing agents. In presence of zinc, hetero-complexes are strongly favored for both protein pairs. We propose that T is conserved in this motif of Cu-transport proteins to promote directional metal transfer toward ATP7B, without formation of energetic sinks. The ability of both Atox1 and WD4 to bind zinc ions may not be a problem in vivo due to the presence of specific transport chains for Cu and Zn ions.

Place, publisher, year, edition, pages
Berlin/Heidelberg: Springer, 2014. Vol. 19, no 6, 1037-1047 p.
Keyword [en]
Atox1, Wilson disease protein, Metal transport, Size exclusion chromatography, Calorimetry
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-92936DOI: 10.1007/s00775-014-1147-0ISI: 000339975100027OAI: oai:DiVA.org:umu-92936DiVA: diva2:747018
Available from: 2014-09-15 Created: 2014-09-09 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Human copper ion transfer: from metal chaperone to target transporter domain
Open this publication in new window or tab >>Human copper ion transfer: from metal chaperone to target transporter domain
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking:

Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein).

My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions.

Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2015. 96 p.
Keyword
copper homeostasis, copper chaperone, Atox1, ATP7B, Wilson disease protein, metal transport, size exclusion chromatography, thermodynamics, isothermal calorimetry
National Category
Inorganic Chemistry Biophysics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-100511 (URN)978-91-7601-203-1 (ISBN)
Public defence
2015-03-27, Lilla Hörsalen, KBC KB3A9, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-03-06 Created: 2015-03-03 Last updated: 2015-03-27Bibliographically approved

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Niemiec, Moritz S.Dingeldein, Artur P. G.Wittung-Stafshede, Pernilla

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