Research ArticleStructural Biology

Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system

See allHide authors and affiliations

Sci. Signal.  10 Apr 2018:
Vol. 11, Issue 525, eaaq0825
DOI: 10.1126/scisignal.aaq0825
  • Fig. 1 Schematic representation of the domain structures of full-length and truncated versions of B. japonicum FixL and FixJ.

    Full-length Bradyrhizobium japonicum FixL comprises two N-terminal Per-Arnt-Sim (PAS) domains, PAS-A and PAS-B, and C-terminal dimerization and histidine phosphotransfer (DHp) and catalytic adenosine triphosphate (ATP)–binding (CA) domains. His200 in the PAS-B domain is critical for binding to heme; His291 in the DHp domain is the site of autophosphorylation; and Asp431-Val467 of the CA domain constitutes the ATP-binding site. Full-length B. japonicum FixJ contains an N-terminal receiver (REC) domain and a C-terminal effector domain that binds to DNA. FixJ is activated by FixL-mediated phosphorylation at Asp55. Structures of the truncated FixL and FixJ proteins FixLPAS-PAS and FixJN used in this study are indicated. The residues that define the boundaries of these domains are noted. Domain structures were generated using the SMART Tool (http://smart.embl-heidelberg.de/).

  • Fig. 2 SEC-SAXS profiles of full-length FixL, FixJ, and FixL-FixJ complexes.

    (A) Log-log plots of x-ray scattering for the met and cyanomet forms of full-length FixL, full-length FixJ, the FixL-FixJ complex, and the FixL-FixJN complex, q = 4πsinθ/λ, where 2θ is the scattering angle and λ is the wavelength of incident x-rays. I(q) is the measured scattered intensity at a given value of 2θ. (B) Corresponding pair distance distribution functions for the indicated proteins and protein complexes. P(r) is the frequency of r intramolecular distances. (C) Dimensionless Kratky plots, which compare the compactness of a protein, for full-length FixL (met form), the truncated FixLPAS-PAS (met form), full-length FixJ, and the full-length FixL-FixJ complex. qRg = q, as defined above, multiplied by the radius of gyration of the protein. A compact protein has a peak maximum at √3 and 1.2 on the abscissa and ordinate, respectively. Movement of the peak further into the positive quartile of the graph indicates unfolding and conformational flexibility. Data were collected on the BL45XU beamline at the SPring-8 synchrotron. n > 3 independent protein preparations and data collections.

  • Fig. 3 Space-filling and pseudoatomic models of full-length FixL.

    SAXS-based models of the met form of full-length FixL showing the overall shape and domain arrangement.

  • Fig. 4 Crystal and SAXS solution structures of full-length FixJ.

    (A) Crystal structure of full-length FixJ (the C2221 data) showing the relative positions of the N-terminal REC (pink), linker (cyan), and C-terminal effector (green) domains. The phosphorylation site, Asp55, is indicated as a stick model with carbon and oxygen atoms indicated in yellow and red, respectively. (B) Comparison of the crystal structures of FixJ in space groups C2221 (blue) and P212121 [yellow (chain A as a representative of chains A to D) and magenta (chain E)]. The view is in the same orientation as (A). (C) Space-filling and pseudoatomic models of full-length FixJ. The ribbon model was colored by temperature factors (b factors). Low and high temperatures are represented in colder and warmer colors, respectively. (D) Comparison of FixJ crystallographic and SAXS models against experimental SAXS data. Experimental SAXS data are shown in black; the calculated scattering curve of the SAXS FixJ model in (C) is shown in red; the calculated scattering curves of the crystal structures of FixJ are shown in other colors as indicated. q = 4πsinθ/λ as defined in Fig. 2.

  • Fig. 5 Space-filling and pseudoatomic models of the FixL-FixJ complex.

    SAXS-based models of the FixL-FixJ complex showing the overall shape, domain arrangement, and mode of complex formation. The C-terminal effector domain of FixJ is not present in the model of the FixL-FixJ complex because it does not form part of the complex interface and is allowed conformational freedom by the linker connecting the N-terminal REC domain to the effector domain.

  • Fig. 6 Schematic representation of the FixL-FixJ TCS.

    Our SAXS results suggest that there are no large changes of the overall shape of full-length FixL upon O2 dissociation from the heme group. However, the orientation of the coiled-coil helices between the heme-containing PAS-B (pink) and DHp (green) domains may change. Such localized structural change could alter the distance between the ATP-binding site in the CA domain (orange) and the autophosphorylation site at His291 in the DHp domain. In the full-length FixL-FixJ complex, a phosphorylation site (Asp55) in the FixJ REC domain approaches His291 of the FixL DHp domain, which mediates phosphotransfer. The C-terminal DNA binding domain of FixJ is connected to the REC domain by a flexible linker, allowing the effector domain to exhibit multiple conformations.

  • Table 1 Phosphotransfer activities of full-length FixL to FixJ by ATP-NADH coupled assay (42).
    State of heme in FixL [activity]Specific activity
    (nmol min−1 mg−1)*
    Deoxy (Fe2+) [active]108 ± 4
    Oxy (Fe2+-O2) [inactive]43 ± 5
    Met (Fe3+) [active]122 ± 6
    Cyanomet (Fe3+-CN) [inactive]12 ± 2

    *The initial rates were obtained from the time courses for the formation of phosphorylated FixJ. These values were calculated from more than three independent time course experiments.

    Oxy FixL was prepared to bind O2 in air to prevent fast autoxidation. The deoxy form accounted for ~30% of the protein in the oxy FixL samples, presumably because of the low affinity of FixL for O2, which was consistent with a previous report (12). Because of the deoxy FixL contaminating the oxy FixL samples, the phosphotransfer activity of the oxy FixL was higher than that of the cyanomet FixL samples, in which all of the proteins were in the inactive state.

    • Table 2 Structural parameters for FixL and FixL-FixJ complexes determined by SEC-SAXS experiments at BL45XU in SPring-8.
      SampleGuinier Rg
      (Å)
      Dmax (Å)Porod
      volume Vp
      3)
      Molecular
      mass from
      Vp (kDa)
      Met FixL49.7 ± 0.1163 ± 4224,220 ±
      3,061
      140.1 ± 1.9
      Cyanomet
      FixL*
      48.4 ± 0.2158 ± 3218,075 ±
      3,693
      136.3 ± 2.3
      Truncated
      FixL
      34.0121100,79063.0
      FixJ22.3 ± 0.266 ± 239,230 ±
      1,115
      24.5 ± 0.7
      Met FixL-FixJ
      complex
      53.1 ± 0.1160 ± 5296,970 ±
      2,478
      185.4 ± 1.5
      Cyanomet
      FixL-FixJ
      complex
      52.1 ± 0.1163 ± 3273,820 ±
      2,253
      171.1 ± 1.4
      Met
      FixL-FixJN
      complex
      48.9 ± 0.1170 ± 3245,753 ±
      2,306
      153.6 ± 1.4

      *The data collection was performed using KCN-containing buffer [40 mM tris-HCl (pH 8.0), 10% (w/v) glycerol, 5 mM potassium cyanide, and 5 mM MgCl2].

      The parameters for truncated FixL were obtained by one measurement. Other samples were analyzed from multiple measurements (n > 3).

      Supplementary Materials

      • www.sciencesignaling.org/cgi/content/full/11/525/eaaq0825/DC1

        Fig. S1. Purification of FixL and FixJ.

        Fig. S2. Optical absorption spectra of full-length FixL.

        Fig. S3. Autophosphorylation and phosphatase activities of FixL.

        Fig. S4. SEC profiles.

        Fig. S5. Space-filling models of FixL alone and in complex with FixJ or FixJN.

        Fig. S6. Pseudoatomic model and SAXS curve of truncated FixL comprising PAS-A, PAS-B, and the coiled-coil region (FixLPAS-PAS).

        Fig. S7. Experimental and simulated SAXS curves of met FixL and the FixL-FixJ complex.

        Fig. S8. SAXS curves and pseudoatomic models of full-length FixL based on comparisons with other HKs.

        Fig. S9. Phosphorylation activities of FixL mutants bearing mutations in the coiled-coil linker.

        Fig. S10. Guinier plots with Pearson residuals for full-length FixL, FixLPAS-PAS, FixL-FixJ, full-length FixJ, and FixJN.

        Table S1. Structural parameters for FixL and FixJ determined by static SAXS experiment at the BL45XU beamline at the SPring-8 synchrotron.

        Table S2. Structural parameters for FixL and FixJ determined by SEC-SAXS at the SWING beamline at the SOLEIL synchrotron.

        Table S3. Crystallographic statistics for full-length FixJ.

      • Supplementary Materials for:

        Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system

        Gareth S. A. Wright, Akane Saeki, Takaaki Hikima, Yoko Nishizono, Tamao Hisano, Misaki Kamaya, Kohei Nukina, Hideo Nishitani, Hiro Nakamura, Masaki Yamamoto, Svetlana V. Antonyuk, S. Samar Hasnain, Yoshitsugu Shiro,* Hitomi Sawai*

        *Corresponding author. Email: yshiro{at}sci.u-hyogo.ac.jp (Y.S.); sawai{at}sci.u-hyogo.ac.jp (H.S.)

        This PDF file includes:

        • Fig. S1. Purification of FixL and FixJ.
        • Fig. S2. Optical absorption spectra of full-length FixL.
        • Fig. S3. Autophosphorylation and phosphatase activities of FixL.
        • Fig. S4. SEC profiles.
        • Fig. S5. Space-filling models of FixL alone and in complex with FixJ or FixJN.
        • Fig. S6. Pseudoatomic model and SAXS curve of truncated FixL comprising PAS-A, PAS-B, and the coiled-coil region (FixLPAS-PAS).
        • Fig. S7. Experimental and simulated SAXS curves of met FixL and the FixL-FixJ complex.
        • Fig. S8. SAXS curves and pseudoatomic models of full-length FixL based on comparisons with other HKs.
        • Fig. S9. Phosphorylation activities of FixL mutants bearing mutations in the coiled-coil linker.
        • Fig. S10. Guinier plots with Pearson residuals for full-length FixL, FixLPAS-PAS, FixL-FixJ, full-length FixJ, and FixJN.
        • Table S1. Structural parameters for FixL and FixJ determined by static SAXS experiment at the BL45XU beamline at the SPring-8 synchrotron.
        • Table S2. Structural parameters for FixL and FixJ determined by SEC-SAXS at the SWING beamline at the SOLEIL synchrotron.
        • Table S3. Crystallographic statistics for full-length FixJ.

        [Download PDF]


        © 2018 American Association for the Advancement of Science

      Navigate This Article