MgATP-Bound and Nucleotide-Free Structures of a Nitrogenase Protein Complex between the Leu 127Δ-Fe-Protein and the MoFe-Protein
| Autor: | James B. Howard, Douglas C. Rees, Hsiu-Ju Chiu, William N. Lanzilotta, John W. Peters, Lance C. Seefeldt, Matthew J. Ryle |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Molybdoferredoxin
Protein Conformation Stereochemistry Crystallography X-Ray Biochemistry Protein Structure Secondary Electron Transport chemistry.chemical_compound Adenosine Triphosphate Protein structure Leucine ATP hydrolysis Oxidoreductase Nitrogenase Nucleotide Binding site Sequence Deletion chemistry.chemical_classification Azotobacter vinelandii Binding Sites biology Chemistry Hydrolysis biology.organism_classification Mutagenesis Site-Directed Adenosine triphosphate |
| Zdroj: | Biochemistry. 40:641-650 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi001645e |
| Popis: | A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from Azotobacter vinelandii (designated the L127Delta-Av2-Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 A resolution and with bound MgATP (introduced by soaking) at 3.0 A resolution. As observed in the structure of the complex between the wild-type A. vinelandii nitrogenase proteins stabilized with ADP.AlF(4-), the most significant conformational changes in the L127Delta complex occur in the Fe-protein component. While the interactions at the interface between the MoFe-protein and Fe-proteins are conserved in the two complexes, significant differences are evident at the subunit-subunit interface of the dimeric Fe-proteins, with the L127Delta-Av2 structure having a more open conformation than the wild-type Av2 in the complex stabilized by ADP.AlF(4-). Addition of MgATP to the L127Delta-Av2-Av1 complex results in a further increase in the separation between Fe-protein subunits so that the structure more closely resembles that of the wild-type, nucleotide-free, uncomplexed Fe-protein, rather than the Fe-protein conformation in the ADP.AlF(4-) complex. The L127Delta mutation precludes key interactions between the Fe-protein and nucleotide, especially, but not exclusively, in the region corresponding to the switch II region of G-proteins, where the deletion constrains Gly 128 and Asp 129 from forming hydrogen bonds to the gamma-phosphate and activating water for attack on this group, respectively. These alterations account for the inability of this mutant to support mechanistically productive ATP hydrolysis. The ability of the L127Delta-Av2-Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding. |
| Databáze: | OpenAIRE |
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