Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

  1. Thomas Spatzal
  2. Kathryn A Perez
  3. James B Howard
  4. Douglas C Rees  Is a corresponding author
  1. Howard Hughes Medical Institute, California Institute of Technology, United States
  2. California Institute of Technology, United States

Abstract

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

Article and author information

Author details

  1. Thomas Spatzal

    Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Kathryn A Perez

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. James B Howard

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Douglas C Rees

    Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States
    For correspondence
    dcrees@caltech.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Wilfred A van der Donk, University of Illinois at Urbana-Champaign, United States

Version history

  1. Received: September 15, 2015
  2. Accepted: December 15, 2015
  3. Accepted Manuscript published: December 16, 2015 (version 1)
  4. Version of Record published: February 3, 2016 (version 2)

Copyright

© 2015, Spatzal et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,437
    Page views
  • 657
    Downloads
  • 108
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Thomas Spatzal
  2. Kathryn A Perez
  3. James B Howard
  4. Douglas C Rees
(2015)
Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor
eLife 4:e11620.
https://doi.org/10.7554/eLife.11620

Share this article

https://doi.org/10.7554/eLife.11620

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Karolina Honzejkova, Dalibor Kosek ... Tomas Obsil
    Research Article

    Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

    1. Biochemistry and Chemical Biology
    Jake W Anderson, David Vaisar ... Natalie G Ahn
    Research Article

    Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.