Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus

Abstract

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Angstrom resolution to R = 0.195 and R(free) = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.

Fig. 1

a Topology diagram of the ferritin from Pyrococcus furiosus (PfFtn). b Ribbon diagram of PfFtn showing the location of the ferroxidase center sites A, B and C (orange spheres) near the center of the four-helical bundle. c Superposition of the PfFtn C^α chain with those of its eight closest homologous structures. In b and c, the molecule colors change from blue to red in the N-terminal to C-terminal direction. b, c Prepared with PyMOL [50]

Fig. 2

Amino acid sequence alignment of PfFtn, the ferritin from Archaeoglobus fulgidus (AfFtn), the ferritin from Escherichia coli (EcFtnA) and human H chain ferritin (HuHF). Asterisks show the residues around the open and E-helix sides of the monomer; A, B and C denote the residues coordinating the iron sites of the ferroxidase center (FC); red bars highlight the main residues located around the threefold channels and gray bars those around the fourfold channels. The alignment was prepared with ClustalW [51]

Fig. 3

Stereoview of a dimer interface in the as-isolated PfFtn structure, showing a difference electron density feature, drawn at the 2.5 map root mean square (RMS) level, which results from a disordered chain of water molecules trapped within a hydrophobic pocket centered around Ile51. This feature is observed in all dimers of all PfFtn structures investigated. The bulk of the PfFtn dimer is represented by its C^α trace (blue-gray). The side chains (including C^α atoms) of the Ile51 residues in both monomers are represented in ball-and-stick mode and are colored gold. The view is down the noncrystallographic twofold symmetry axis of the dimer, looking towards the inside of the PfFtn 24-mer. The figure was prepared with DINO [52]

Fig. 4

Ribbon diagrams of the PfFtn and AfFtn 24-mers. I View down a threefold axis, showing one “fundamental hexamer.” II View rotated by approximately 90° about a vertical axis, showing the different hexamer arrangement, which leads to the appearance of large pores in AfFtn. The figure was prepared with PyMOL [50]

Fig. 5

Profile view of the threefold and fourfold channels in PfFtn, AfFtn, EcFtnA and HuHF. The exterior of the shell lies on the left side and the inner cavity on the right side of each cartoon as shown schematically in a. The red residues correspond to acidic Glu and Asp residues and the blue ones to positive Lys, Arg and His residues, and highlight the arrangement of positive and negative amino acids along the channels. The figure was prepared with PyMOL [50]

Fig. 6

View of the FC down the PfFtn subunit from the E-helix side. The three metal sites are annotated as A, B and C. Metal occupation of sites B and C is observed only upon crystal soaking in Zn or Fe. The dashed lines correspond to the coordination geometries described in Table 5. The figure was prepared with PyMOL [50]

Fig. 7

Details of the FC of a selected monomer in the structures of as-isolated, Fe-loaded, Fe-soaked and Zn-soaked PfFtn crystals. a Final 2|F_o| − |F_c| electron-density map for the as-isolated PfFtn, contoured at 1.2 map RMS. b Final 2|F_o| − |F_c| electron-density map for the Fe-soaked PfFtn, contoured at 1.2 map RMS. c Final 2|F_o| − |F_c| electron-density map for the Zn-soaked PfFtn, contoured at 1.2 map RMS. d Anomalous Fourier map for the Fe-loaded PfFtn, contoured at 3.0 map RMS. e Anomalous Fourier map for the Fe-soaked PfFtn, contoured at 3.0 map RMS. f Anomalous Fourier map for the Zn-soaked PfFtn using the peak data, contoured at 3.0 map RMS. g Labeled view of the residues and metal sites in the Fe-soaked FC. h Anomalous Fourier map for the Zn-soaked PfFtn using the low-energy remote data, contoured at 3.0 map RMS. i Dispersive Fourier map for the Zn-soaked PfFtn, contoured at 2.8 map RMS. The anomalous electron-density maps were calculated using as amplitudes the anomalous difference coefficients obtained from each dataset, and the phases (rotated by 90°) obtained from the respective final structure refinement, except for the “Fe-loaded” data, for which the phases were taken from the “as-isolated” refinement. The Zn-dispersive Fourier map was calculated by first scaling together the peak and low-energy remote datasets with CCP4 SCALEIT [16] and then using as coefficients the difference F(low-energy remote)  − F(peak) and the phases from the final “Zn-soak” refinement. a–c show that the occupation of site B in the as-isolated crystal is negligible; d shows a small residual occupancy of site B in the Fe-loaded structure and no evidence of site C occupancy; e shows a site B occupancy clearly lower than those of sites A and C in the Fe-soaked structure; f, h and i show that in the Zn-soaked structure, part of the originally present Fe in site A has been displaced to site C by the Zn ions, that site B is occupied by Zn only, and that site C is very likely occupied by Fe only. All panels were drawn with the same orientation. In all panels except g the bulk of PfFtn monomer is represented as a gray tube onto which the side chains (including C^α atoms) of the FC residues have been overlaid in ball-and-stick representation (carbon atoms blue-gray, oxygen atoms red, nitrogen atoms blue, iron atoms pink and zinc atoms cyan). In g, only the residues near the FC are represented in ball-and-stick mode, and are labeled for easier identification of the residues mentioned in the text. The figure was prepared with DINO [52]

Fig. 8

The Fe(III)–O–Fe(II) FC electron paramagnetic resonance (EPR) signal of 6 μM PfFtn 24-mer, titrated to 130 mV. The decrease in the amplitude of the signal depending on incubation time and the presence of crystallization solution in the sample illustrates the stability of the center. Prior to EPR measurement, the three samples tested were incubated, respectively, for 1 day after loading with iron (blue line), for 2 months (red line) and for 2 months in the presence of a crystallization solution, 2 M ammonium sulfate (black line)