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Kahra, Dana
Publications (6 of 6) Show all publications
Hoernke, M., Mohan, J., Larsson, E., Kahra, D., Westenhoff, S., Lundmark, R. & Schwieger, C. (2017). Determining membrane bound protein structures by infrared reflection-absorption spectroscopy. Paper presented at 19th IUPAB Congress / 11th EBSA Congress, JUL 16-20, 2017, British Biophys Soc, Edinburgh, SCOTLAND. European Biophysics Journal, 46, S161-S161
Open this publication in new window or tab >>Determining membrane bound protein structures by infrared reflection-absorption spectroscopy
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2017 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S161-S161Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Springer, 2017
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-143670 (URN)000416406201062 ()
Conference
19th IUPAB Congress / 11th EBSA Congress, JUL 16-20, 2017, British Biophys Soc, Edinburgh, SCOTLAND
Note

Supplement: 1, Meeting Abstract: P-177

Available from: 2018-01-16 Created: 2018-01-16 Last updated: 2018-06-09Bibliographically approved
Hoernke, M., Mohan, J., Larsson, E., Blomberg, J., Kahra, D., Westenhoff, S., . . . Lundmark, R. (2017). EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation. Proceedings of the National Academy of Sciences of the United States of America, 114(22), E4360-E4369
Open this publication in new window or tab >>EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 22, p. E4360-E4369Article in journal (Refereed) Published
Abstract [en]

The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

Keywords
EHD2, caveolae, membrane-reshaping protein, membrane-bound protein structure, infrared flection-absorption spectroscopy
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-136326 (URN)10.1073/pnas.1614066114 (DOI)000402296700009 ()28223496 (PubMedID)
Available from: 2017-06-22 Created: 2017-06-22 Last updated: 2018-06-09Bibliographically approved
Kahra, D., Kovermann, M. & Wittung-Stafshede, P. (2016). The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1. Biophysical Journal, 110(1), 95-102
Open this publication in new window or tab >>The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1
2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 1, p. 95-102Article in journal (Refereed) Published
Abstract [en]

Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, K-D, of similar to 10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.

National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-114887 (URN)10.1016/j.bpj.2015.11.016 (DOI)000367783900032 ()26745413 (PubMedID)
Available from: 2016-04-27 Created: 2016-01-29 Last updated: 2018-06-07Bibliographically approved
Kahra, D., Mondol, T., Niemiec, M. S. & Wittung-Stafshede, P. (2015). Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro. Protein peptide letters, 22(6), 532-538
Open this publication in new window or tab >>Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro
2015 (English)In: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 22, no 6, p. 532-538Article in journal (Refereed) Published
Abstract [en]

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.

Place, publisher, year, edition, pages
Bentham Science, 2015
Keywords
Atox1, Copper chaperone, fluorescence microscopy, fluorescence spectroscopy, transcription factor
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-105277 (URN)000355192800008 ()25962064 (PubMedID)
Available from: 2015-06-22 Created: 2015-06-22 Last updated: 2018-06-07Bibliographically approved
Petzoldt, S., Kahra, D., Kovermann, M., Dingeldein, A. P., Niemiec, M. S., Ådén, J. & Wittung-Stafshede, P. (2015). Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro. Biometals, 28(3), 577-585
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, p. 577-585Article 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
Keywords
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: 2018-06-07Bibliographically approved
Hoernke, M., Mohan, J., Larsson, E., Blomberg, J., Kahra, D., Daumke, O., . . . Lundmark, R.TP driven stabilization of a membrane bound open confirmation of the ATPase EHD2 restrains caveolae dynamics.
Open this publication in new window or tab >>TP driven stabilization of a membrane bound open confirmation of the ATPase EHD2 restrains caveolae dynamics
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-120758 (URN)
Available from: 2016-05-20 Created: 2016-05-20 Last updated: 2018-06-07
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