2fwh: Difference between revisions
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[[Image:2fwh.gif|left|200px]]<br /><applet load="2fwh" size=" | [[Image:2fwh.gif|left|200px]]<br /><applet load="2fwh" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="2fwh, resolution 0.99Å" /> | caption="2fwh, resolution 0.99Å" /> | ||
'''atomic resolution crystal structure of the C-terminal domain of the electron transfer catalyst DsbD (reduced form at pH7)'''<br /> | '''atomic resolution crystal structure of the C-terminal domain of the electron transfer catalyst DsbD (reduced form at pH7)'''<br /> | ||
==Overview== | ==Overview== | ||
Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to | Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD. | ||
==About this Structure== | ==About this Structure== | ||
2FWH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with IOD and PEG as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Protein-disulfide_reductase Protein-disulfide reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.8.1.8 1.8.1.8] Full crystallographic information is available from [http:// | 2FWH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=IOD:'>IOD</scene> and <scene name='pdbligand=PEG:'>PEG</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Protein-disulfide_reductase Protein-disulfide reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.8.1.8 1.8.1.8] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2FWH OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Protein-disulfide reductase]] | [[Category: Protein-disulfide reductase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Boeckmann, R | [[Category: Boeckmann, R A.]] | ||
[[Category: Capitani, G.]] | [[Category: Capitani, G.]] | ||
[[Category: Glockshuber, R.]] | [[Category: Glockshuber, R.]] | ||
[[Category: Grauschopf, U.]] | [[Category: Grauschopf, U.]] | ||
[[Category: Gruetter, M | [[Category: Gruetter, M G.]] | ||
[[Category: Rozhkova, A.]] | [[Category: Rozhkova, A.]] | ||
[[Category: Stirnimann, C | [[Category: Stirnimann, C U.]] | ||
[[Category: IOD]] | [[Category: IOD]] | ||
[[Category: PEG]] | [[Category: PEG]] | ||
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[[Category: thioredoxin-like]] | [[Category: thioredoxin-like]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:25:51 2008'' | ||
Revision as of 18:25, 21 February 2008
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atomic resolution crystal structure of the C-terminal domain of the electron transfer catalyst DsbD (reduced form at pH7)
OverviewOverview
Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD.
About this StructureAbout this Structure
2FWH is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as Protein-disulfide reductase, with EC number 1.8.1.8 Full crystallographic information is available from OCA.
ReferenceReference
High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study., Stirnimann CU, Rozhkova A, Grauschopf U, Bockmann RA, Glockshuber R, Capitani G, Grutter MG, J Mol Biol. 2006 May 5;358(3):829-45. Epub 2006 Feb 28. PMID:16545842
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