| 1. |
- Andersson, Magnus, et al.
(författare)
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Transport Pathway in Cu+ P-Type ATPases
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 106:2, s. 427A-427A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 2. |
- Grønberg, Christina, et al.
(författare)
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Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport
- 2016
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 111:11, s. 2417-2429
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Tidskriftsartikel (refereegranskat)abstract
- Cu+-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu+-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu+ entry using molecular-dynamics simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu+ delivery. Mutational analyses and simulations in the presence and absence of Cu+ predict that the ion-entry path involves two ion-binding sites: one transient Met148-Cys382 site and one intramembranous site formed by trigonal coordination to Cys384, Asn689, and Met717. The results reconcile earlier biochemical and x-ray absorption data and provide a molecular understanding of ion entry in Cu+-transporting P-type ATPases.
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| 3. |
- Liu, Xiangyu, et al.
(författare)
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Overproduction of PIB-Type ATPases.
- 2016
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029. ; 1377, s. 29-36
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals.
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| 4. |
- Mattle, Daniel, et al.
(författare)
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A sulfur-based transport pathway in Cu+-ATPases
- 2015
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Ingår i: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 16:6, s. 40-728
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Tidskriftsartikel (refereegranskat)abstract
- Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB -type Cu(+)-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu(+) across cellular membranes. Crystal structures of a copper-free Cu(+)-ATPase are available, but the mechanism of Cu(+) recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu(+)-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu(+) is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway.
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| 5. |
- Nass, Karol, et al.
(författare)
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Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams
- 2015
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Ingår i: Journal of Synchrotron Radiation. - 0909-0495 .- 1600-5775. ; 22:2, s. 225-238
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Tidskriftsartikel (refereegranskat)abstract
- Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe–4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe–4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.
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| 6. |
- Sitsel, Oleg, et al.
(författare)
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Crystallization of P-type ATPases by the High Lipid-Detergent (HiLiDe) Method.
- 2016
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Ingår i: P-Type ATPases. - New York, NY : Springer New York. - 1064-3745. - 9781493931781 ; 1377, s. 413-420
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Determining structures of membrane proteins remains a significant challenge. A technique utilizing high lipid-detergent concentrations ("HiLiDe") circumvents the major bottlenecks of current membrane protein crystallization methods. During HiLiDe, the protein-lipid-detergent ratio is varied in a controlled way in order to yield initial crystal hits, which may be subsequently optimized by variation of the crystallization conditions and/or utilizing secondary detergents. HiLiDe preserves the advantages of classical lipid-based methods, yet is compatible with both the vapor diffusion and batch crystallization techniques. The method has been applied with particular success to P-type ATPases.
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| 7. |
- Sitsel, Oleg, et al.
(författare)
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Structure and Function of Cu(I)- and Zn(II)-ATPases
- 2015
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 54:37, s. 5673-5683
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Tidskriftsartikel (refereegranskat)abstract
- Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing the molecular differences that allow transport of these two distinct metal types, and discuss possible future directions of research in the field.
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| 8. |
- Wang, Kaituo, et al.
(författare)
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Structure and mechanism of Zn2+-transporting P-type ATPases
- 2014
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 514:7523, s. 518-
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Tidskriftsartikel (refereegranskat)abstract
- Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis(1). In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements(2,3). Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.P-i) of ZntA from Shigella sonnei, determined at 3.2 angstrom and 2.7 angstrom resolution, respectively. The structures reveal a similar fold to Cu+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn2+ ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2.P-i state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn2+ release as a built-in counter ion, as has been proposed for H+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between P-IB-type Zn2+-ATPases and P-III-type H+-ATPases and at the same time show structural features of the extracellular release pathway that resemble P-II-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase(4,5) (SERCA) and Na+, K+-ATPase(6). These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.
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