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MgATP-Bound and Nucleotide-Free Structures of a Nitrogenase Protein Complex between the Leu 127Δ-Fe-Protein and the MoFe-Protein

Chiu, Hsiu-Ju and Peters, John W. and Lanzilotta, William N. and Ryle, Matthew J. and Seefeldt, Lance C. and Howard, James B. and Rees, Douglas C. (2000) MgATP-Bound and Nucleotide-Free Structures of a Nitrogenase Protein Complex between the Leu 127Δ-Fe-Protein and the MoFe-Protein. Biochemistry, 40 (3). pp. 641-650. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20140729-102140505

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Abstract

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 L127Δ-Av2−Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 Å resolution and with bound MgATP (introduced by soaking) at 3.0 Å 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 L127Δ 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 L127Δ-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 L127Δ-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 L127Δ 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 γ-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 L127Δ-Av2−Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://pubs.acs.org/doi/abs/10.1021/bi001645ePublisherArticle
http://dx.doi.org/10.1021/bi001645eDOIArticle
ORCID:
AuthorORCID
Rees, Douglas C.0000-0003-4073-1185
Additional Information:© 2001 American Chemical Society. Received July 14, 2000; Revised Manuscript; Received November 7, 2000. This work was supported by NIH Grant GM45162 (D.C.R. and J.B.H.), NSF Grant MCB 9722937 (L.C.S.), and NIH Postdoctoral Fellowship GM18142 (J.W.P.). The rotation camera facility at the Stanford Synchrotron Radiation Laboratory is supported by the Department of Energy Office of Basic Sciences and the NIH Biomedical Technology Program, Division of Research Resources.
Funders:
Funding AgencyGrant Number
NIHGM45162
NSFMCB 9722937
NIHGM18142
Department of Energy (DOE) Office of Basic SciencesUNSPECIFIED
Issue or Number:3
Record Number:CaltechAUTHORS:20140729-102140505
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20140729-102140505
Official Citation:MgATP-Bound and Nucleotide-Free Structures of a Nitrogenase Protein Complex between the Leu 127Δ-Fe-Protein and the MoFe-Protein. Hsiu-Ju Chiu, John W. Peters, William N. Lanzilotta, Matthew J. Ryle, Lance C. Seefeldt, James B. Howard, and, and Douglas C. Rees Biochemistry 2001 40 (3), 641-650
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:47553
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:29 Jul 2014 17:54
Last Modified:03 Oct 2019 06:54

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