Introduction 1 2 3 4 5 6 9 2 3 2 Archaeoglobus fulgidus 2 Halobacterium salinarum 10 10 11 Listeria innocua 12 2 Pyrococcus furiosus P. furiosus 2 2 2 P. furiosus Escherichia coli 13 Herein we present the 3D structure of PfFtn, refined at 2.75-Å resolution. In contrast to the previously reported first structure of an archaeal AfFtn, the functional PfFtn 24-mer has the typical 432 point-group symmetry observed in other ferritins and BFRs. There are no four large pores such as were found in AfFtn. Instead, the characteristic threefold and fourfold channels are present in PfFtn. P. furiosus Thermotoga maritima The FC stability was analyzed by electron paramagnetic resonance (EPR). Without further experiments, the lack of FC iron sites B and C in nonsoaked PfFtn crystals obtained does not allow any conclusions supporting or contradicting the FC as a stable catalytic cofactor. Materials and methods Protein production and crystallization  13 N N 14 −1 14 ε −1 −1 4 2 2 4 2 2 2 4 X-ray data collection Cryoprotecting conditions consisted of briefly dipping the crystals into a modified crystallization solution containing 20% glycerol in the case of the as-crystallized and Fe-loaded crystals, and 25% glycerol in the case of the Fe-soaked and Zn-soaked crystals, prior to flash-freezing at −173 °C in a nitrogen-gas stream, using an Oxford Cryosystems low-temperature device. 15 16 1 Table 1 X-ray diffraction data collection and processing statistics Beamline ESRF BM-14 ESRF BM-14 ESRF BM-14 ESRF BM-14 ESRF BM-14 Detector MAR 225 CCD MAR 225 CCD MAR 225 CCD MAR 225 CCD MAR 225 CCD Crystal As-isolated Fe-loaded 4 4 4 Wavelength (Å) 1.7712 1.7340 1.4585 1.2810 1.305 Space group  C 1 C 1 C 1 C 1 C 1 Unit cell (Å) a 255.00 254.32 254.30 255.37 255.69 b 341.42 343.16 342.88 342.06 342.36 c 265.52 266.26 266.22 265.99 266.21 Resolution range (Å) 58.2–2.75 (2.90–2.75) 63.2–2.95 (3.11–2.95) 42.0–2.80 (2.95–2.80) 42.9–2.80 (2.95–2.80) 42.9–2.80 (2.95–2.80) Observations 1,685,078 (151,184) 903,956 (85,605) 1,011,149 (102,079) 1,082,567 (111,068) 1,086,719 (111,952) Unique reflections 295,335 (42,009) 237,583 (32,389) 280,407 (38,583) 279,537 (37,607) 280,474 (37,777) Completeness (overall) (%) 99.7 (97.8) 98.1 (92.2) 99.1 (94.1) 98.7 (91.6) 98.7 (91.9) Redundancy 5.7 (3.6) 3.8 (2.6) 3.6 (2.6) 3.9 (3.0) 3.9 (3.0) R merge 0.085 (0.300) 0.113 (0.532) 0.081 (0.354) 0.075 (0.288) 0.077 (0.344) I σ I 16.5 (3.1) 10.8 (1.6) 8.2 (2.0) 8.8 (2.5) 8.6 (1.9) Anomalous completeness (%) 98.0 (88.3) 89.2 (71.1) 94.4 (64.8) 97.7 (85.4) 97.6 (85.5) Anomalous redundancy 2.9 (1.9) 2.0 (1.5) 1.8 (1.6) 1.9 (1.5) 2.0 (1.5) R anom 0.043 (0.204) 0.078 (0.414) 0.058 (0.294) 0.063 (0.252) 0.059 (0.288) B overall 2 53.3 62.7 60.5 54.3 58.5 parentheses Structure determination of as-isolated and Fe-loaded PfFtn 14 17 14 14 18 19 20 F o Structure refinement of as-isolated PfFtn 21 22 23 B B 2 R R free R R free R free 24 F o F c F o F c 2 B F o F c F o F c Table 2 Pyrococcus furiosus Crystal As-isolated, as-crystallized Fe-soaked Zn-soaked Resolution (Å) 56.8–2.75 42.0–2.80 42.8–2.80 No. of reflections in work set/test set 280,375/14,914 266,173/14,187 265,356/14,134 R work R free 0.195/0.247 0.197/0.249 0.201/0.251 Number of atoms  Protein—total number of atoms 49,824 49,824 49,824  Protein—atoms with zero occupancy 788 718 720  Fe/Zn atoms 36 108 108  Water molecules (sulfate ions) 791 (41) 413 (26) 162 (44) B 2  Protein main chain (side chain) 32.6 (34.6) 38.0 (39.9) 30.9 (32.7)  Fe/Zn atoms 57.5 53.2 36.9  Water molecules (sulfate ions) 26.5 (67.1) 29.0 (76.8) 21.9 (74.0) Root mean square deviations from ideal values  Bond lengths (Å) 0.014 0.014 0.015  Bond angles (°) 1.44 1.46 1.48  Mean positional error (Å) 0.20 0.22 0.22 PROCHECK Ramachandran analysis  Total no. analyzed residues 5,544 5,544 5,544  Residues in core regions, no. (%) 5,255 (94.79) 5,241 (94.53) 5,239 (94.50)  Residues in allowed regions, no. (%) 277 (5.00) 285 (5.14) 289 (5.21)  Residues in generous regions, no. (%) 11 (0.20) 10 (0.18) 9 (0.16)  Residues in disallowed regions, no. (%) 1 (0.02) 8 (0.14) 7 (0.13) G  Mean (range) dihedral 0.00 (−0.13 to 0.07) −0.04 (−0.23 to 0.03) −0.04 (−0.21 to 0.09)  Mean (range) covalent 0.38 (0.32–0.45) 0.38 (0.29–0.46) 0.36 (0.26–0.44)  Mean (range) overall 0.15 (0.07–0.19) 0.12 (0.00–0.18) 0.12 (0.01–0.17) 25 26 ϕ φ ϕ φ 27 Structure determination and refinement of the Fe-soaked and Zn-soaked PfFtn crystals 22 F o F c F o F c 28 Discussion” 2 23 B B R R free 2 25 26 ϕ φ ϕ φ 23 B B R R free 2 25 26 ϕ φ ϕ φ R R free R free 24 h k l R free R work 29 R free 28 F o F c F o F c F o F c F o F c 27 Results and discussion Structure of the PfFtn monomer 30 1 Fig. 1 a Pyrococcus furiosus b orange spheres c α b c blue red b c 50  2 E. coli Fig. 2 PfFtn Archaeoglobus fulgidus AfFtn Escherichia coli EcFtnA HuHF Asterisks A B C red bars gray bars 51  2 The FC 1 2 A surface hydrophobic pocket at the PfFtn dimer interface Desulfovibrio desulfuricans 31 3 32 Fig. 3 α blue-gray α gold 52  Subunit structure comparison http://www.ebi.ac.uk/msd-srv/ssm 33 3 α 1 Q 3 Table 3 Proteins with known 3D structure most closely homologous to PfFtn PDB ID Protein name Source organism Chain length Identity (%) Identity SS-aligned (%) Q a RMSD (Å) 1vlg Ferritin (TM1128)  Thermotoga maritima 176 55 55.3 0.92 0.49 1s3q Ferritin Archaeoglobus fulgidus 173 50 48.2 0.94 0.53 1krq Ferritin Campylobacter jejuni 167 39 39.2 0.81 1.02 1eum Ferritin ECFTNA Escherichia coli 165 38 38.4 0.86 0.90 1r03 Mitochondrial ferritin Homo sapiens  182 40 30.6 0.68 1.55 2fha H ferritin K86Q mutant Homo sapiens  183 37 29.4 0.62 1.98 1mfr M ferritin Rana catesbeiana 176 36 29.6 0.68 1.67 1rcg L ferritin Rana catesbeiana 173 32 25.0 0.61 2.03 PDB SS RMSD a Structure of the PfFtn 24-mer and comparison with AfFtn 2 4 2 Fig. 4 I II 50  Threefold and fourfold channels 5 5 5 Fig. 5 left side right side a red blue 50  9 34 36 34 2+ 37 5 5 The FC 1 38 4 E. coli Table 4 The ferroxidase center in PfFtn and its homologous crystallized ferritins PDB ID Protein name Source organism Resolution (Å) FC type Metal site occupation – Ferritin Pyrococcus furiosus 2.75 FtnA  1s3q Ferritin Archaeoglobus fulgidus 2.10 FtnA a  1vlg Ferritin (TM1128)  Thermotoga maritima 2.00 FtnA A (Fe) 1krq Ferritin Campylobacter jejuni 2.70 FtnA b 1eum Ferritin ECFTNA Escherichia coli 2.05 FtnA c  1r03 Mitochondrial ferritin Homo sapiens sapiens 1.70 HuHF – 2fha H ferritin K86Q mutant Homo sapiens sapiens 1.90 HuHF – 1mfr M ferritin Rana catesbeiana 2.80 HuHF – 1rcg L ferritin Rana catesbeiana 2.20 – – FC a 2 b c 1 2+ 3+ 5 6 F o F c 7 Table 5 Coordination geometry statistics of the ferroxidase center in PfFtn a Distance (Å)  b b b A 2.24 (0.09) [2.00–2.46] 2.21 (0.08) [2.00–2.47] 2.18 (0.07) [2.01–2.32] A 2.26 (0.11) [2.09–2.47] 2.24 (0.09) [2.09–2.55] 2.22 (0.07) [2.02–2.38] A 2.12 (0.09) [1.97–2.41] 2.28 (0.09) [2.12 – 2.47] 2.21 (0.08) [2.08 – 2.40] A B  3.02 (0.19) [2.62–3.63] 2.93 (0.18) [2.54–3.49] A C  7.41 (0.10) [7.18–7.63] 7.51 (0.11) [7.30–7.77] B  2.21 (0.12) [1.93–2.45] 2.16 (0.12) [1.92–2.50] B  2.34 (0.14) [2.12 – 2.70] 2.40 (0.10) [2.18 – 2.65] B  2.87 (0.16) [2.47–3.14] 2.81 (0.20) [2.41–3.21] B C  6.29 (0.20) [5.81–6.77] 6.25 (0.15) [5.93–6.55] C  2.11 (0.08) [1.92 – 2.36]  2.29 (0.11) [2.13 – 2.56] C  2.06 (0.09) [1.87–2.26] 2.31 (0.16) [1.84–2.53] C  2.31 (0.10) [2.12–2.58] 2.02 (0.10) [1.81–2.26] C  1.89 (0.08) [1.72–2.04] 1.89 (0.09) [1.66–2.07] a M b parentheses Fig. 6 A B C B C dashed lines 5 50  Fig. 7 a F o c b F o F c c F o F c d e f g h i 16 F F a c d e f h i g gray tube α blue-gray red blue pink cyan g 52  7 7 7 7 7 2 1 FC stability 38 D. desulfuricans 31 39 39 8 8 5 Fig. 8 blue line red line black line  38 Thermostability 40 46 47 48 13 6 P. furiosus 6 6 Table 6 Hydrogen bonds and salt bridges in the PfFtn 24-mer and its closest structural homologues, calculated with HBPLUS PDB ID a b MM MS SS c Intramolecular Intermolecular Intramolecular Intermolecular Intramolecular Intermolecular Intramolecular Intermolecular – Pyrococcus furiosus 4,630 3,377 24 439 72 544 174 172 67 1vlg Thermotoga maritima 4,767 3,237 30 540 117 633 210 189 120 1s3q Archaeoglobus fulgidus 4,457 2,788 24 412 80 453 176 114 134 1krq Campylobacter jejuni 4,632 3,360 24 648 72 384 144 72 72 1eum Escherichia coli 4,720 3,320 24 836 32 412 96 60 4 MM MS SS a parentheses b c 49 Campylobacter jejuni 6 6 6 Conclusions In this study, we reported the crystal structure of PfFtn. High sequence similarity between PfFtn, TmFtn and AfFtn suggests a separate group of thermostable ferritins. PfFtn is the second archaeal ferritin structure to be reported, following that of AfFtn. In spite of the high sequence identity (50%) between these two proteins, their quaternary structures are significantly different. PfFtn exhibited the 432 symmetry commonly found in other known ferritins, whereas AfFtn showed the 23 symmetry similar to dodecameric DPS proteins. The 23 symmetry of the AfFtn 24-mer resulted in four triangular pores with an approximate size of 45 Å, making large openings in the AfFtn shell. Therefore, the 23 symmetry of the AfFtn 24-mer remains an exception among ferritins, and further research is required to explain such an unusual conformation. Three iron sites were observed in the FC of PfFtn monomer. Site A was present in the as-crystallized protein and sites B and C were observed only upon crystal soaking with either Fe or Zn ions. Combining these results with our measurements of the FC EPR signal prior to crystallization, where samples were incubated in air for 1 day and 2 months, and for 2 months in 2 M ammonium sulfate (the crystallization solution), we conclude that the lack of iron atoms at the B site is very likely due to their complexation by ammonium sulfate, and that the absence of these sites in the as-crystallized protein cannot be taken per se as evidence of a transient nature of the FC, in PfFtn and other ferritins. The possible iron entry and exit routes of PfFtn, the threefold and fourfold channels, are similar to those from bacterial ferritins in the sense that the threefold channel is less hydrophilic and the fourfold channel is more polar than the channels of mammalian H and L chain ferritins. T. maritima