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In vitro thermodynamic dissection of human copper transfer from chaperone to target protein
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
2012 (engelsk)Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 5, s. e36102-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

sted, utgiver, år, opplag, sider
2012. Vol. 7, nr 5, s. e36102-
HSV kategori
Identifikatorer
URN: urn:nbn:se:umu:diva-56355DOI: 10.1371/journal.pone.0036102ISI: 000305349800032PubMedID: 22574136OAI: oai:DiVA.org:umu-56355DiVA, id: diva2:533715
Tilgjengelig fra: 2012-06-14 Laget: 2012-06-14 Sist oppdatert: 2018-06-08bibliografisk kontrollert
Inngår i avhandling
1. Human copper ion transfer: from metal chaperone to target transporter domain
Åpne denne publikasjonen i ny fane eller vindu >>Human copper ion transfer: from metal chaperone to target transporter domain
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Umeå: Umeå Universitet, 2015. s. 96
Emneord
copper homeostasis, copper chaperone, Atox1, ATP7B, Wilson disease protein, metal transport, size exclusion chromatography, thermodynamics, isothermal calorimetry
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-100511 (URN)978-91-7601-203-1 (ISBN)
Disputas
2015-03-27, Lilla Hörsalen, KBC KB3A9, Umeå Universitet, Umeå, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2015-03-06 Laget: 2015-03-03 Sist oppdatert: 2018-06-07bibliografisk kontrollert

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