Structural modeling of a novel membrane-bound globin-coupled sensor in Geobacter sulfurreducens

Abstract

Globin-coupled sensors (GCS) usually consist of three domains: a sensor/globin, a linker, and a transmitter domain. The globin domain (GD), activated by ligand binding and/or redox change, induces an intramolecular signal transduction resulting in a response of the transmitter domain. Depending on the nature of the transmitter domain, GCSs can have different activities and functions, including adenylate and di-guanylate cyclase, histidine kinase activity, aerotaxis and/or oxygen sensing function. The gram-negative delta-proteobacterium Geobacter sulfurreducens expresses a protein with a GD covalently linked to a four transmembrane domain, classified, by sequence similarity, as GCS (GsGCS). While its GD is fully characterized, not so its transmembrane domain, which is rarely found in the globin superfamily. In the present work, GsGCS was characterized spectroscopically and by native ion mobility-mass spectrometry in combination with cryo-electron microscopy. Although lacking high resolution, the oligomeric state and the electron density map were valuable for further rational modeling of the full-length GsGCS structure. This model demonstrates that GsGCS forms a transmembrane domain-driven tetramer with minimal contact between the GDs and with the heme groups oriented outward. This organization makes an intramolecular signal transduction less likely. Our results, including the auto-oxidation rate and redox potential, suggest a potential role for GsGCS as redox sensor or in a membrane-bound e^-/H⁺ transfer. As such, GsGCS might act as a player in connecting energy production to the oxidation of organic compounds and metal reduction. Database searches indicate that GDs linked to a four or seven helices transmembrane domain occur more frequently than expected.

Keywords: AfGcHK, Anaeromyxobacter sp. Fw109-5 GcHK; AsFRMF, Ascaris suum FRMF-amide receptor; AvGReg, Azotobacter vinilandii Greg; BpGReg, Bordetella pertussis Greg; BsHemAT, Bacillus subtilis HemAT; CCS, collision cross section; CIU, collision-induced unfolding; CMC, critical micelle concentration; CV, cyclic voltammetry; CeGLB26, Caenorhabditis elegans globin 26; CeGLB33, Caenorhabditis elegans globin 33; CeGLB6, Caenorhabditis elegans globin 6; DDM, n-dodecyl-β-d-maltoside; DPV, differential pulse voltammetry; EcDosC, Escherichia coli Dos with DGC activity; FMRF, H-Phe-Met-Arg-Phe-NH2 neuropeptide; GCS, globin-coupled sensor; GD, globin domain; GGDEF, Gly-Gly-Asp-Glu-Phe motive; Gb, globin; Geobacter sulfurreducens; GintHb, hemoglobin from Gasterophilus intestinalis; Globin-coupled sensor; GsGCS, Geobacter sulfurreducens GCS; GsGCS162, GD of GsGCS; IM-MS, ion mobility-mass spectrometry; LmHemAC, Leishmania major HemAC; MaPgb, Methanosarcina acetivorans protoglobin; MtTrHbO, Mycobacterium tuberculosis truncated hemoglobin O; NH4OAc, ammonium acetate; OG, n-octyl-β-d-glucopyranoside; PDE, phosphodiesterase; PcMb, Physether catodon myoglobin; PccGCS, Pectobacterium carotivorum GCS; PsiE, phosphate-starvation-inducible E; RR, resonance Raman; SCE, saturated calomel electrode; SHE, standard hydrogen electrode; SaktrHb, Streptomyces avermitilis truncated hemoglobin-antibiotic monooxygenase; SwMb, myoglobin from sperm whale; TD, Transmitter domain; TmD, Transmembrane domain; Transmembrane domain; Transmembrane-coupled globins; mNgb, mouse neuroglobin.

Graphical abstract

Fig. 1

UV/Vis spectra of GsGCS in different heme iron oxidation and coordination states. The spectra correspond to the as-purified ferric (blue), ferrous deoxy (red), and CO-bound ferrous (black) form. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Resonance Raman spectra of GsGCS in different iron oxidation and coordination states. High (a) and low frequency (b) resonance Raman spectra corresponding to as-purified ferric (i), ferrous deoxy (ii), and CO-bound ferrous (iii) forms of GsGCS. The applied laser power was 1 mW (iii) and 100 mW (i and ii).

Fig. 3

Mass spectra of GsGCS¹⁶² under native and denaturing conditions. The native spectrum (a) shows two species corresponding to monomeric and dimeric GsGCS¹⁶². The detected mass reflects the protein species including the noncovalent prosthetic heme group (black dot). The denaturing spectrum (b) shows only monomeric GsGCS¹⁶² upon denaturation, indicating the non-covalent character of the dimerization. The extended charge state distribution relative to the native spectrum highlights the stepwise unfolding of the protein from compact (C) to intermediate (I) and extended (E) structures, displayed by three Gaussian-like peak distributions and ion mobility. Denaturation also led to the release of the noncovalently-bound heme group (616.29 m/z).

Fig. 4

Native MS spectrum of GsGCS solubilized in OG micelles. The spectrum shows two charge state distributions corresponding to trimeric (117,948 Da) and tetrameric (157,186 Da) GsGCS. The detected mass reflects the protein including three and four noncovalent prosthetic heme groups for trimers and tetramers, respectively. Minor satellite peaks indicate the loss of one heme group (*, 616 Da) and likely a lipid bound to the complex (+, 690 Da).

Fig. 5

CIU of dimeric (+12) GsGCS¹⁶² and tetrameric (+28) GsGCS in different iron oxidation/coordination states. CIU plots of dimeric (+12) GsGCS¹⁶² (a) show the unfolding transition (32.5 V collision energy voltage) for each species with almost no difference between them. CIU plots of tetrameric (+28) GsGCS (b) however show large differences among the different states. The as-purified Fe(III) ferric and the Fe(II) CO-bound form unfold at 120 and 110 V collision energy voltage, respectively. Contrary, the ferrous deoxy species show a much higher unfolding collision energy voltage (160 V), indicating a more stable form of this complex.

Fig. 6

Electron Micrograph and 2D classification of full-length GsGCS. (a) Electron micrograph of negatively stained full-length GsGCS in OG detergent. Protein is bright against a darker background. The scale bar equals 50 nm. (b) 2D classification: class averaged from 21,414 cryo-particles, the box size equals 135 × 135 Å².

Fig. 7

3D models of full-length GsGCS in OG. (a) Structures obtained by applying of C3 (yellow) and C4 (cyan) symmetry are illustrated from different angles. (b) Description of the various domains. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Structural model of GsGCS-PsiE tetramer. Side (a) and top (b) view of the tetrameric model obtained after fitting four monomeric GsGCS-PsiE subunits into the cryo-EM envelope. The model shows four loosely connected globin domains with outward-oriented heme groups, providing easy access. Each monomeric subunit is shown in a different color. Helices of the globin domain (A–H), the linker (L) and the transmembrane domain (Tm1–Tm4) are labeled accordingly in one subunit.

Fig. 9

Close-up view of the globin domain. Helix arrangement and heme group of the globin domains of both the model (a) and the X-ray structure GsGCS¹⁶² (pdb 2W31) (b). Comparing the X-ray structure with the model clearly highlight differences in the interactions between their globin domains. The model of the full-length protein, including the TmD, no longer forms a tightly connected 4-α-helical bundle between G/H helices. Instead, the G/H helices of one monomer are facing the E/F helices of the next subunit by going clockwise. The heme groups (orange) are orientated to the outer edge of the molecule enabling access for potential ligands. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)