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Human copper ion transfer: from metal chaperone to target transporter domain
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Pernilla Wittung-Stafshede)
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 [en]
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: urn:nbn:se:umu:diva-100511ISBN: 978-91-7601-203-1 (print)OAI: oai:DiVA.org:umu-100511DiVA: diva2:792419
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
List of papers
1. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein
Open this publication in new window or tab >>In vitro thermodynamic dissection of human copper transfer from chaperone to target protein
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 5, e36102- p.Article in journal (Refereed) 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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-56355 (URN)10.1371/journal.pone.0036102 (DOI)000305349800032 ()22574136 (PubMedID)
Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2017-12-07Bibliographically approved
2. Enthalpy-entropy compensation at play in human copper ion transfer
Open this publication in new window or tab >>Enthalpy-entropy compensation at play in human copper ion transfer
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C1XXC2 motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cysto-Ala variants, we demonstrate that Cu-dependent hetero-protein complexes require the presence of C1 but not C2. Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild-type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.

Keyword
copper chaperone, Atox1, Wilson disease protein, metal transport, size exclusion chromatography, thermodynamics, calorimetry
National Category
Biophysics Biochemistry and Molecular Biology Inorganic Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-100509 (URN)
Note

accepted at Scientific Reports, no DOI yet.

Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2015-03-04Bibliographically approved
3. T versus D in the MTCXXC motif of copper transport proteins plays a role in directional metal transport
Open this publication in new window or tab >>T versus D in the MTCXXC motif of copper transport proteins plays a role in directional metal transport
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
Keyword
Atox1, Wilson disease protein, Metal transport, Size exclusion chromatography, Calorimetry
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-92936 (URN)10.1007/s00775-014-1147-0 (DOI)000339975100027 ()
Available from: 2014-09-15 Created: 2014-09-09 Last updated: 2017-12-05Bibliographically approved
4. Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein
Open this publication in new window or tab >>Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein
Show others...
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 42, 13038-13050 p.Article in journal (Refereed) Published
Abstract [en]

Although strictly regulated, pH and solute concentrations in cells may exhibit temporal and spatial fluctuations. Here we study the effect of such changes on the stability, structure, and dynamics in vitro and in silico of a two-domain construct (WD56) of the fifth and sixth metal-binding domains of the copper transport protein, ATP7B (Wilson disease protein). We find that the thermal stability of WD56 is increased by 40 °C when increasing the pH from 5.0 to 7.5. In contrast, addition of salt at pH 7.2 decreases WD56 stability by up to 30 °C. In agreement with domain-domain coupling, fractional copper loading increases the stability of both domains. HSQC chemical shift changes demonstrate that, upon lowering the pH from 7.2 to 6, both His in WD6 as well as the second Cys of the copper site in each domain become protonated. MD simulations reveal increased domain-domain fluctuations at pH 6 and in the presence of high salt concentration, as compared to at pH 7 and low salt concentration. Thus, the surface charge distribution at high pH contributes favorably to overall WD56 stability. By introducing more positive charges by lowering the pH, or by diminishing charge-charge interactions by salt, fluctuations among the domains are increased and thereby overall stability is reduced. Copper transfer activity also depends on pH: delivery of copper from chaperone Atox1 to WD56 is more efficient at pH 7.2 than at pH 6 by a factor of 30. It appears that WD56 is an example where the free energy landscapes for folding and function are linked via structural stability.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-71579 (URN)10.1021/jp402415y (DOI)23675861 (PubMedID)
Available from: 2013-06-04 Created: 2013-06-04 Last updated: 2017-12-06Bibliographically approved
5. Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro
Open this publication in new window or tab >>Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro
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2015 (English)In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, no 3, 577-585 p.Article in journal (Refereed) Published
Abstract [en]

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P-1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Place, publisher, year, edition, pages
Springer, 2015
Keyword
Human copper transport, Atox1, Copper chaperone for superoxide dismutase, (SOD), Size exclusion chromatography, Proton-NMR
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-100334 (URN)10.1007/s10534-015-9832-1 (DOI)000354273900014 ()25673218 (PubMedID)
Available from: 2015-03-01 Created: 2015-03-01 Last updated: 2017-12-04Bibliographically approved

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