1m1y
From Proteopedia
(New page: 200px<br /><applet load="1m1y" size="450" color="white" frame="true" align="right" spinBox="true" caption="1m1y, resolution 3.2Å" /> '''Chemical Crosslink of...) |
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| - | [[Image:1m1y.gif|left|200px]]<br /><applet load="1m1y" size=" | + | [[Image:1m1y.gif|left|200px]]<br /><applet load="1m1y" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1m1y, resolution 3.2Å" /> | caption="1m1y, resolution 3.2Å" /> | ||
'''Chemical Crosslink of Nitrogenase MoFe Protein and Fe Protein'''<br /> | '''Chemical Crosslink of Nitrogenase MoFe Protein and Fe Protein'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The transient formation of a complex between the component Fe- and | + | The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure. |
==About this Structure== | ==About this Structure== | ||
| - | 1M1Y is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Azotobacter_vinelandii Azotobacter vinelandii] with CA, HCA, CFM, CLF and SF4 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nitrogenase Nitrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.18.6.1 1.18.6.1] Full crystallographic information is available from [http:// | + | 1M1Y is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Azotobacter_vinelandii Azotobacter vinelandii] with <scene name='pdbligand=CA:'>CA</scene>, <scene name='pdbligand=HCA:'>HCA</scene>, <scene name='pdbligand=CFM:'>CFM</scene>, <scene name='pdbligand=CLF:'>CLF</scene> and <scene name='pdbligand=SF4:'>SF4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nitrogenase Nitrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.18.6.1 1.18.6.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1M1Y OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Nitrogenase]] | [[Category: Nitrogenase]] | ||
[[Category: Protein complex]] | [[Category: Protein complex]] | ||
| - | [[Category: Chiu, H | + | [[Category: Chiu, H J.]] |
[[Category: Einsle, O.]] | [[Category: Einsle, O.]] | ||
| - | [[Category: Howard, J | + | [[Category: Howard, J B.]] |
| - | [[Category: Rees, D | + | [[Category: Rees, D C.]] |
[[Category: Schmid, B.]] | [[Category: Schmid, B.]] | ||
[[Category: Willing, A.]] | [[Category: Willing, A.]] | ||
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[[Category: protein interaction]] | [[Category: protein interaction]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:50:39 2008'' |
Revision as of 11:50, 21 February 2008
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Chemical Crosslink of Nitrogenase MoFe Protein and Fe Protein
Overview
The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.
About this Structure
1M1Y is a Protein complex structure of sequences from Azotobacter vinelandii with , , , and as ligands. Active as Nitrogenase, with EC number 1.18.6.1 Full crystallographic information is available from OCA.
Reference
Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure., Schmid B, Einsle O, Chiu HJ, Willing A, Yoshida M, Howard JB, Rees DC, Biochemistry. 2002 Dec 31;41(52):15557-65. PMID:12501184
Page seeded by OCA on Thu Feb 21 13:50:39 2008
Categories: Azotobacter vinelandii | Nitrogenase | Protein complex | Chiu, H J. | Einsle, O. | Howard, J B. | Rees, D C. | Schmid, B. | Willing, A. | Yoshida, M. | CA | CFM | CLF | HCA | SF4 | Chemical crosslink | Nitrogen fixation | Protein interaction
