High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin Structural biology Renzo Johansson1, Eduard Torrents2, Daniel Lundin3, Janina Sprenger1, Margareta Sahlin3, Britt-Marie Sjoberg3 and Derek T Logan1 ă Department of Biochemistry and Structural Biology, Lund University, Sweden Cellular Biotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain Department of Molecular Biology and Functional Genomics, Stockholm University, Sweden Keywords crystal structure; flavin mononucleotide; flavodoxin; NrdI; ribonucleotide reductase Correspondence D T Logan or B.-M Sjoberg, Department ă of Biochemistry and Structural Biology, Lund University, Box 124, S-221 00 Lund; Department of Biochemistry of Molecular Biology and Functional Genomics, Stockholm University, S-106 91 Stockholm, Sweden Fax: +46 46 222 4692; +46 16 64 88 Tel: +46 46 222 1443; +46 16 41 50 E-mail: derek.logan@mbfys.lu.se; britt-marie sjoberg@molbio.su.se Website: http://www.mps.lu.se; http://www.molbio.su.se Database Structural data for oxidized and reduced NrdI are available in the Protein Data Bank under the accession numbers 2XOD and 2XOE (Received 12 July 2010, revised 10 August 2010, accepted 18 August 2010) The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria NrdI interacts with the class Ib radical generating protein NrdF It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the ˚ oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 A, respectively These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10 The implications of these observations for class Ib ribonucleotide reductase function are discussed doi:10.1111/j.1742-4658.2010.07815.x Introduction The ribonucleotide reductase (RNR) system is essential for genome replication and repair in all free-living organisms, comprising the enzymes that carry out the first committed step of synthesis of the building blocks of DNA, namely the conversion of ribonucleotides to deoxyribonucleotides RNRs have a highly diverse set of radical generation, storage and transfer strategies, and are divided into three classes on this basis [1–3] Class I RNRs have a strict requirement for oxygen, whereas the class II enzymes are indifferent to the Abbreviations MR, molecular replacement; PDB, Protein Data Bank; RNR, ribonucleotide reductase; RNRdb, Ribonucleotide Reductase Database FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS 4265 NrdI – an unusual flavodoxin R Johansson et al degree of aerobicity and the class III RNRs are strictly anaerobic Class I RNRs are further divided into class Ia and Ib based on differences in operon structure, allosteric activity regulation and domain structure [4–6], and recent phylogenetic studies demonstrate that class Ib is restricted to the bacterial kingdom [7] The class Ia RNRs use a di-iron-oxo metal centre to generate a stable tyrosyl radical in protein NrdB (R2), which is then reversibly tranported through a conserved radical transfer pathway to the active site on protein NrdA (R1) when required for catalysis [1] The class Ib homologue of NrdA is NrdE (or R1E) and the class Ib protein NrdF (or R2F) is equivalent to NrdB in class Ia Class Ia RNRs are allosterically regulated with regard to both overall activity and substrate specificity [2,3] Class Ib RNRs are not regulated for overall activity [5], and there is some ambiguity concerning the nature of their metal centres The first, manganese-containing, RNR from Corynebacterium ammoniagenes [8] was later shown to be a class Ib RNR that was also functional in an Fe-containing form [9,10] Recently, the class Ib RNR from Escherichia coli was also shown to be enzymatically active, both as a Mn- and as a Fe-containing enzyme [11,12] NrdI is a small flavodoxin-like protein whose gene is found in all organisms where class Ib ribonucleotide reductases (RNR) are present It was first identified in the mid-1990s as part of the nrdEF gene cluster in Escherichia coli and Salmonella typhimurium [13] In these enterobacteria, it was found to code for a small protein of 136 amino acids with a molecular mass of 15.3 kDa, normally forming an nrdHIEF operon structure Subsequently, NrdI was shown to be involved in the activity of class Ib RNR [14], having a stimulatory effect on NrdEF activity A decade later, NrdI was demonstrated to be essential for class Ib RNR activity in Streptococcus pyogenes [15], which contains two redundant and simultaneously expressed class Ib gene clusters: NrdHEF and NrdF*I*E* The latter system was not active in enzymatic assays in vitro but, in complementation experiments, NrdF*I*E* was able to restore lost class Ib RNR activity in a temperaturesensitive E coli strain This led to the first proposal that NrdI could be essential for maintenance of class Ib RNR activity [15] Recently, a thorough investigation of the potential roles of NrdI in function of class Ib RNR in E coli has been carried out Two non-mutually exclusive hypotheses have been proposed [11,12] In the first scenario, NrdI is suggested to be involved in rescue of active NrdF proteins whose FeIII-FeIII-Tyr° centres have been reduced by one electron to produce the inactive FeIII-FeIII-Tyr (met) form This rescue would 4266 be effected by the injection of two electrons in rapid succession into the FeIII-FeIII centre to produce a reduced FeII-FeII centre, which would then react with molecular oxygen according to the well-characterized assembly pathway [16] to regenerate active NrdF Importantly, NrdI was shown to differ significantly in its redox properties from previously characterized Flds, which typically alter the redox potentials for the ox ⁄ sq and sq ⁄ hq couples of their FMN cofactors in such a way that the flavin group becomes a one-electron reductant Flds normally stabilize near stoichiometric amounts of the neutral sq form of FMN by shifting the redox couple Esq ⁄ hq from )172 mV for free FMN [17] to between )370 and )450 mV for the bound form [18] and Eox ⁄ sq from )238 mV for the free form to between )50 and )220 mV for the bound form [18] By contrast, the protein environment of E coli NrdI maintains the redox potentials of the two couples at very similar values, namely Eox ⁄ sq = )264 mV and Esq ⁄ hq = )255 mV, respectively [11] In this way, FMN bound to E coli NrdI may be made capable of injecting two electrons in rapid succession into NrdF The interaction with NrdI was also shown to be specific for NrdF because no effect was seen on the class Ia NrdB protein Given the ambiguity as to the nature of the redoxactive metal species in class Ib RNRs, an alternative scenario has been investigated in which NrdI is involved in the assembly of an active MnIII-MnIII-Tyr° cofactor in E coli NrdF [12] The two proteins were found to form a tight complex during nickel–nitrilotriacetic acid affinity chromatography Aerobic incubation of fully-reduced NrdI with MnII-reconstituted NrdF led to the formation of active NrdF with 0.25 tyrosyl radicals per dimer This was suggested to occur through the reaction of the MnII centre with two equivalents of HO2) produced by two successive oneelectron reductions of O2 by NrdIhq bound to NrdF By contrast, aerobic incubation of NrdF reconstituted with FeII in the presence of NrdI led to a species with only 13% of the specific activity, although it had 0.2 tyrosyl radicals per dimer It was thus proposed that NrdI is involved in the assembly of a MnIII-MnIII-Tyr° cofactor in E coli and that this is the true cofactor in vivo However, this hypothesis does not exclude the possibility that the cofactor is FeIII–FeIII-Tyr° under some growth conditions, and that NrdI could be involved in maintenance of the cofactor under these circumstances E coli expresses a class Ia RNR during aerobiosis that cannot be substituted for by its chromosomally encoded class Ib RNR This is in contrast to many bacterial species that are dependent upon their class Ib FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS R Johansson et al NrdI – an unusual flavodoxin RNR for aerobic growth It is therefore of interest to study the structural and functional properties of class Ib RNR from organisms such as the Bacillus cereus group and, in the present study, we present the structure of NrdI from the human pathogen Bacillus anthracis, baNrdI [19] Although the baNrdI protein is highly similar to the B cereus protein recently reported in partially photoreduced forms [20], the previous study concentrated on the structural effects on the flavin of photoreduction during data collection Given the functional disparities between NrdI and normal Flds, it is important to study the structural basis for NrdI function In the present study, we present the crystal structures of baNrdI in the oxidized and chemically-reduced semiquinone forms NrdI is shown to have an unusually compact Fld fold, defining a new structural class within the Fld family The electrostatic potential surface of baNrdI is shown to be strikingly different to that of Flds A bioinformatic analysis of a large number of NrdI sequences shows that this effect is general; indeed, on average, NrdI proteins are significantly basic and their electrostatic and redox properties can be expected to vary to a surprising degree is Fld from Desulfovibrio desulfuricans [dsFld; Protein Data Bank (PDB) code: 3F6R] [22], with a rmsd of ˚ 2.4 A for 113 alignable Ca atoms out of 117 in baNrdI and 147 in dsFld Very similar statistics are obtained for a wide variety of Flds from diverse organisms, whether short-chain or long-chain However, B anthracis NrdI is 30 residues shorter than a typical short chain Fld and displays a more compact fold The truncations occur principally on the side of baNrdI furthest from the flavin binding site Helix a1 is shorter by five residues (seven versus 13), strand b2 by four residues (three versus seven) and strand b3 by four residues (five versus nine) (Fig and Table S1) In addition, the loops between a1 and b2; a3 and b4; and a4 and b5 are shortened compared to dsFld Analysis of 199 NrdI sequences extracted from the Ribonucleotide Reductase Database (RNRdb) [7] (Fig S1) shows that NrdIs are fairly homogeneous in length, with a median value of 141 and a SD of 13 There is no division into short- and long-chain variants as there is for Fld The minimum structural core, with shortest loops, is apparently represented by the Corynebacterium striatum sequence at 109 amino acids (Fig S1) Variations in length are essentially limited to the termini and the loops between a1 and b2, between b2 and b3, and the loop (residues 42–49) that interacts with the flavin moiety of FMN The first two variable loops are distant from FMN The variable length of this ‘‘40s loop’’ is discussed below By contrast, beyond the FMN-binding loop, the NrdI structure is extremely well conserved (Fig S1), with no significant insertions or deletions The electron density for the FMN cofactor is excellent in both structures, and the high resolution of the oxidized form allows confirmation of the protonation Results The crystal structure of baNrdI has been solved to ˚ 0.96 A resolution with the FMN cofactor in its oxi˚ dized state and to 1.4 A with chemically-reduced FMN B anthracis NrdI is unambiguously a member of the Fld superfamily The fold consists of a fivestranded parallel b-sheet flanked by two a-helices on each side (Fig 1) However, a search using the dali server [21] indicates that NrdI is a structural outlier within the Fld family The closest structural neighbour A B 90s loop 70s loop 50s loop 40s loop Fig (A) Overall structure of NrdI The cartoon is coloured as a rainbow from blue at the N-terminus to red at the C-terminus to emphasize the topology For clarity, helix is semi-transparent The FMN cofactor is shown in a stick representation Lengths of the secondary structure elements are given in Table S1 (B) Structure of the most structurally homologous standard flavodoxin, the short chain protein from D desulfuricans (PDB ID 3F6R), for comparison The representation is as shown in (A) Prepared using PYMOL β5 β5 α1 α2 α4 β4 β3 β1 β2 FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS α4 β4 α1 β3 α2 β1 β2 4267 NrdI – an unusual flavodoxin R Johansson et al state In electron density maps calculated without inclusion of explicit hydrogen atoms on the cofactor, the difference electron density can clearly be seen at 2.5 r for many of the aliphatic hydrogen atoms of the cofactor (Fig 2A) By contrast, no hydrogen atoms can be seen on N5, confirming the oxidized state of FMN The phosphate group of FMN is bound by the A E98 D105 70s loop α1 3.4 2.9 W74 D76 D83 2.8 3.2 3.1 G44 F45 40s loop T43 90s loop E120 B D92 W90 D59 D58 M56 D57 E62 E65 50s loop D98 E101 E63 Fig (A) Electron density for the FMN cofactor and the 40s loop ˚ The grey mesh shows a rA-weighted 2|Fo| ) |Fc| map to 0.96 A calculated using SHELXL and contoured at 1.2 r around the FMN cofac˚ tor and the 40s loop Residues within A of FMN that make interactions with it are shown as thin lines Particularly relevant side chains, including all acidic side chains in the view, are shown as sticks Hydrogen bonds are shown as dashed lines The green ˚ mesh shows a similarly calculated Fo ) Fc map to 1.1 A resolution contoured at 2.5 r For calculation of this map, hydrogen atoms were included for the protein but omitted from the cofactor (B) The flavodoxin from C beijerinckii in its oxidized state (PDB code: 4NLL) in the same orientation as baNrdI shown in (A) The representation is identical to that shown in (A) The overall details of FMN binding are very similar to baNrdI; however, note the preponderance of acidic residues in the vicinity of the FMN binding site Prepared using PYMOL 4268 N-terminus of a1 and the preceding P-loop The flavin moiety of FMN binds in a pocket formed by loops at the C-terminal ends of b-strands and (loops 42–49 and 71–79, respectively), known in Fld as the W and Y loops, or the 50s and 90s loops [23] We refer to these as the 40s and 70s loops, respectively, in baNrdI The flavin moiety is sandwiched between Trp74 on one face and the side chain of Thr42 and the main chain atoms of residues 42–44 on the other (Fig 2A) The isoalloxazine ring is completely buried and anchored through seven hydrogen bonds between its carbonyl (O2, O4) and amide (N3) groups and main-chain carbonyl and amide groups in the 40s and 70s loops By contrast, the dimethylbenzene ring is solvent-exposed The flavin binding pocket is capped by Phe45, whose side chain lies perpendicular to the flavin moiety and also makes an edge-on interaction to the stacking Trp74 The flavin environment in NrdI is considerably less negatively charged than in Fld For example, Clostridium beijerinckii Fld (cbFld) has a net charge of )14, whereas in baNrdI it is only )4 (in the present study the net protein charges always refer to the protein component only) Figure 2B shows the preponderance of acidic side chains in the vicinity of the flavin in cbFld, including two in the 50s loop itself There are ˚ three acidic residues within A, and a further four ˚ Overall, 26 negative charges are compenwithin 10 A sated by only 12 positive charges By contrast, in baNrdI, 18 negative charges are compensated for by 14 positive ones The closest of these to the flavin, Asp76 ˚ in the 70s loop and Asp83 in helix a4, are A distant from the flavin (Fig 2A) This has a remarkable effect on the electrostatic energy landscape of NrdI compared to Fld (Fig 3) To test the generality of this observation, we carried out an analysis of the length, amino acid composition and calculated isoelectric point of a representative set of 199 NrdI sequences extracted from the RNRdb A parallel analysis of 38 manually reviewed flavodoxin sequences from the UniRef100 database (http://www.uniprot.org) was performed for comparison, confirming that NrdI differs strongly from flavodoxins with respect to pI The median pI for NrdI sequences is 9.0 with a SD of 1.7 (Fig 4A) However, the spread in values is wide, ranging from 4.2 for Eubacterium biforme to 10.4 for NrdI1 of S pyogenes M1 Net charges vary remarkably, from )15 to +15 The pI distribution is approximately bimodal, with a major peak at pH 9.0–9.5 and a broader peak at pH 5.0–5.5 By contrast, the 38 representative flavodoxin sequences that have been analyzed are much more homogeneous in pI: the median value is 4.5 with a SD of only 0.6 (Fig 4B) FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS R Johansson et al A NrdI – an unusual flavodoxin B Fig Electrostatic potentials for (A) baNrdI and (B) C beijerinckii flavodoxin The potentials at the solvent accessible surface were calculated using APBS [53] and mapped onto the molecular surface using PYMOL The colour scale in both panels runs from deep red at )5 kTe)1 to blue at +5 kTe)1 The FMN molecule is shown in a space-filling representation The molecular surface is semi-transparent and a grey cartoon of each molecule is shown for orientation The direction of view is into the side of the flavin plane Prepared using PYMOL 60 A 50 Flavin photoreduction as a result of X-ray exposure 40 30 20 10 B 25 20 15 10 largest conformational change in the protein is a peptide flip between residues 44 and 45, resulting in the orientation of the Gly44 carbonyl group towards N5, which is protonated in the neutral sq radical form (Fig 5A) This peptide flip is accompanied by a slight rearrangement of the whole loop from Thr42 to Asn47 Interestingly, Thr42 undergoes a small shift, and difference density appears at a level of approximately r between its side chain and the flavin ring (Fig 5B) This density is also present at approximately r in the maps of the chemically-reduced B cereus NrdI [20] as generated by the Electron Density Server [24] (http://eds.bmc.uu.se/eds/), although the authors responsible for this entry did not interpret it The ˚ density is too close to Thr42 (1.7 A to Oc) to be a water molecule, although it could be a loosely coordinated metal ion There is also a rotamer change in Thr43 and a slight movement of Phe45 upon flavin reduction This confirms that the conformational response of NrdI to reduction is very similar to that observed in flavodoxins [18,25–27] 9 9 9 9 9 9 9 9 9 9 –3 0–4 0–4 0–5 0–5 0–6 0–6 0–7 0–7 0–8 0–8 0–9 0–9 –10 50 5 5 5 4 5 6 7 8 9 0.0 Fig Histograms of the distributions of predicted isoelectric point for (A) NrdI and (B) Fld sequences The sequences are colour-coded according to the phylum of the source organisms The pI groups to which the NrdIs with determined structures belong are labelled: Bant, B anthracis; Bcer, B cereus; Bsub, B subtilis; Ecol, E coli Produced using Google Docs (http://docs.google.com/) Reduced baNrdI-FMN The crystal structure of fully reduced baNrdI-FMN was obtained by chemical reduction of crystals of oxidized baNrdI using 500 mm sodium dithionite The Røhr et al [20] recently noted the need to take into consideration the effects of radiation damage on the geometry of flavin cofactors when analysing structures where data were collected using synchrotron X-ray sources A significant distortion of the flavin was noted in both NrdIox and NrdIsq from B cereus after estimated radiation doses of and 10 MGy, respectively Quantum mechanics simulations of the flavin geometry in the protein context coupled to resonance Raman experiments on the crystals suggested that both NrdIox and NrdIsq had been reduced by one electron during X-ray exposure, such that the flavins were now in the FMN°) and FMNH) states respectively With this in mind, we investigated the effect of radiation damage in the almost identical B anthracis system Using suitable parameters for the I911-3 (NrdIox) and I911-5 (NrdIsq) beamlines at MAX-lab, Lund, Sweden), we arrived at a dose estimate of approximately 2–4 MGy for baNrdIox and 5–6 MGy for baNrdIsq, using the software raddose [28] In the last cycle of refinement, no restraints were used in the refinement of the FMN geometry for oxidized baNrdI The ‘butterfly angle’ between the flavin ring planes is 5.7°, which compares well with the value of 4.8° reported for the oxidized B cereus protein after photoreduction, indicating that one-electron reduction has also occurred in baNrdI The resolution of the sq form was not sufficiently high to allow unrestrained refinement, and so a similar comparison is not FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS 4269 NrdI – an unusual flavodoxin R Johansson et al A B FMN 3.2 W74 3.4 O2 2.8 S69 2.4 2.8 2.1 T42 F45 T43 G44 Fig (A) Conformational change upon chemical reduction of baNrdI to the semiquinone state The grey mesh shows a rA-weighted ˚ 2|Fo| ) |Fc| map to 1.4 A resolution calculated using REFMAC5 and contoured at 1.2 r around the FMN cofactor and the 40s loop Reduced baNrdI is shown in green and the 40s loop of the oxidized form is shown in blue for comparison Hydrogen bonds between FMN and the 40s loop are shown as dashed lines (B) The strong difference density that appears between the flavin and Thr42 in the crystal structure of reduced baNrdI, which is also present in bcNrdI (Fig S2) A 2|Fo| ) Fc| map contoured at 1.0 r and an |Fo| ) |Fc| map contoured at 3.0 r are shown in grey and green, respectively The height of the difference map peak is approximately r A marker atom has been placed in the electron density to show the distances to potential coordinating atoms in the vicinity Prepared using PYMOL meaningful However, distortion of the flavin geometry as a result of accumulated photoreduction during data collection can be seen in the anisotropic B-factors of the flavin atoms in both oxidized and sq forms (Fig 6) With the isoallazine ring being fixed by its interactions with the protein, the flavin distortion is concentrated on the dimethylbenzene ring, which has greater freedom to distort from planar geometry Discussion The crystal structures of NrdI from B anthracis have been solved in two functional states: in complexes with oxidized and semiquinone FMN The structures reveal that NrdI is unambiguously a member of the Fld superfamily, although it has the most compact fold of a Fld seen to date, being shorter than the average short-chain Fld It can thus be considered to define a new family within the Fld superfamily The NrdI family is not divided into short- and long-chain subfamilies: the region in and around the final strand b5, where the insertion defining the long-chain Flds occurs, is extremely highly conserved with regard to secondary structure in NrdI sequences (Fig S1) Despite being an outlier in the Fld family, the FMNbinding regions are more conserved than the rest of the structure Correctly-folded baNrdI for functional and structural studies could only be obtained by including FMN in the growth medium (in our case LB medium) at a concentration of 60 lm, when overexpressed in 4270 E coli In the absence of FMN NrdI was misfolded and produced irreversibly in inclusion bodies Without FMN, NrdI from S typhimurium, C ammoniagenes, B anthracis and Deinococcus radiodurans also form inclusion bodies during heterologous overexpression in E coli (E Torrents, unpublished results) The observation is also in agreement with previously published studies on E coli NrdI, in which significant quantities of functional protein could only be obtained by refolding from inclusion bodies in the presence of FMN [11] A general requirement for FMN in NrdI folding would contrast with the behaviour of traditional Flds The dependence of Fld folding on FMN has been studied, and the binding of FMN to native apo-Fld was found to constitute the last step [29] The autonomous formation of native apo-Fld is essential during holo-Fld folding, and FMN does not act as a nucleation site for folding FMN can be removed from Fld by acid treatment [30], despite affinity in the subnanomolar range [29], also resulting in a stable apoprotein Conformational differences between apo- and holo-Flds are small and confined to the 50s and 90s loops [31,32] Further experiments are required to establish whether NrdI has a general requirement for FMN for correct folding during overexpression NrdI is remarkably less negatively charged than normal flavodoxins The major function of the protein environment in flavodoxins is modification of the redox potentials FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS R Johansson et al A B Fig Depiction of the anisotropic movements of atoms in the FMN molecule as represented by their anisotropic B-factors (A) ˚ ˚ NrdIox, 0.96 A resolution, refined using SHELXL (B) NrdIsq, 1.4 A, refined using REFMAC5 The anisotropic B-factors are represented by thermal ellipsoids at 50% probability The B-factors are coloured ˚ ˚ from dark blue at 5.0 A2 to bright red at 12.8 A2 in (A) and from ˚ ˚ 7.7 A2 to 18.7 A2 using the same colour scheme in (B) Prepared using PYMOL ox ⁄ sq and sq ⁄ hq couples from the rather similar values of )172 mV and )238 mV, respectively, in the free flavin in FMN [17] to the widely different values of )50 to 260 mV and )370 to )450 mV, respectively, for the bound form [18] Thereby, flavodoxins are made into effective one-electron donors The effects on redox potentials occur primarily through (a) stabilization of the sq form via a hydrogen bond between the N5 atom and a carbonyl group in the 50s loop [18,25,26,33–36] and (b) destabilization of the negatively-charged hq form (FMNH)) through the lack of solvation in the protein environment coupled to highly negative electrostatic field, which reduces the protein’s association rate with the hq form of FMN [18,36,37] Replacement of acidic residues by neutral or positively-charged ones tends to increase the sq ⁄ hq potential [18,38,39], although theoretical studies have shown that compensatory (de)protonation effects on other charged residues can make the effect difficult to predict [37] By contrast, modification of the conformational properties of the 50s loop affects the ox ⁄ sq potential to a greater degree [26,35,36] The remarkably similar redox potentials of the ox ⁄ sq and sq ⁄ hq couples in E coli NrdI (ecNrdI) have NrdI – an unusual flavodoxin been attributed to a lesser destabilization of the sq form, FMNH), than what is normally the case in flavodoxins [11] The structure of baNrdI confirms this hypothesis, and our bioinformatic analysis extends it, with few exceptions, to the whole NrdI family The conformational changes observed between the oxidized and reduced states are limited to a peptide flip between residues 44 and 45 and a small rearrangement of the 40s loop The high similarity of these changes to those observed in Fld from several species strongly suggests that the unusual redox potentials of NrdI are governed more by protein electrostatics than by specific hydrogen bonds or other direct interactions with the flavin However, the wide spread in predicted pI values for NrdI sequences is unexpected The data point to the interesting possibility of two distinct functional groups with acidic and basic characters, respectively, which obviously will have quite different effects on the potentials of the FMN redox couples Alternatively, the charge variation in NrdI may be correlated with a similar variation in the electrostatic properties of the respective NrdF proteins, in particular at the interaction area The reason for the wide spread in predicted pI is not obvious Figure 4A shows the distribution colour-coded by taxonomy It can be seen that NrdIs from the a- and c-proteobacteria belong almost exclusively to the high-pI group, whereas the firmicutes and actinobacteria occupy a wide range of pI values Several organisms in the latter two groups, including some Bacillus species, encode more than one class Ib operon, whereas the proteobacteria all encode only one In general, the NrdI pI values differ substantially within organisms encoding two different nrdI genes A phylogenetic tree of 91 representative NrdI sequences (Fig S3) shows that the NrdI phylogeny is not significantly different from that based on NrdF sequences [19] or on 16S rRNA, although actinobacteria and firmicutes, with two class Ib operons, generally have NrdIs divided into two separate clusters However, the presence of a sequence in a genome provides no information regarding if and when the protein is expressed, and so further experiments are required to establish the reason for the wide spread in electrostatic properties in an otherwise structurally conserved family E coli NrdI, with pI = 9.4, belongs to the major group of NrdI sequences with basic character, whereas baNrdI, with pI = 5.4, belongs to the minor, acidic group These proteins also differ in their ability to stabilize an FMN sq radical: a maximum of 28% can be detected in ecNrdI, whereas, in baNrdI, the amount is up to 60% (M Sahlin & B.-M Sjoberg, unpublished ă results) Interestingly, this correlates with the predicted pIs: that of baNrdI (net charge )4) is much closer to FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS 4271 NrdI – an unusual flavodoxin R Johansson et al that of a normal flavodoxin than that of ecNrdI (net charge +4) In E coli NrdI, a neutral sq radical is produced when NrdI is titrated anaerobically with dithionite [11] In the presence of NrdF, whether in the apo- or Mn-containing forms, an anionic sq radical is produced instead This behaviour was described as being potentially more similar to that of flavoprotein oxidases than flavodoxins [12] However, the stabilization of an anionic sq form would be favoured by the presence of a positivelycharged protein residue in the vicinity of the N1-C2-O2 atoms [40], as in glycolate oxidase [41] No such residue can be found in baNrdI and, indeed, the formation of anionic sq should be disfavoured by the overall negative charge of the protein This points to influence of NrdF on the flavin environment in the NrdF ⁄ I complex, although the flavin N1-C2-O2 atoms would remain inaccessible to residues from NrdF in the complex, unless NrdF induces a conformational change in the 90s loop to expose the flavin We have identified the possibility that a metal ion is trapped close to the N1-C2-O2 locus in chemically-reduced baNrdI and bcNrdI, in the space made available by the conformational rearrangement in the 40s loop induced by the peptide flip of Gly44 (Fig 5B) This may help to compensate the negative charge in an anionic sq form chromatography [12] The capping of the FMN binding site by Phe45 appears to be specific for Bacillus and a few other species, and is correlated with the exceptionally short loop found in baNrdI This residue is Gly in almost all other NrdI sequences Thus, the extended loop may close off the flavin binding site by folding back over the core of the protein When the present paper was in revision, the crystal structures of the complex between NrdI and NrdF from E coli in the oxidized and hq forms were published [43] This work confirmed our prediction that the variable 40s loop will contribute to the different affinities of NrdI for NrdF in various organisms The equivalent loop of NrdI in the E coli complex forms an important part of the molecular interface and undergoes more significant conformational changes upon reduction than in the Bacillus species, although this may be influenced by its interactions with NrdF In summary, we have identified the NrdI protein as a structural outlier within the flavodoxin family, having a significantly more compact fold, although the structure close to the flavin binding site is more conserved The 40s loop is an important site of sequence variation in the NrdI family A very wide distribution in predicted pI values has been identified, which may imply different functional roles for NrdI in different organisms The FMN-binding loop is a major site of sequence variation in NrdI Materials and methods NrdI sequences vary in length from 103 to 188 amino acids Intriguingly, one of the most variable loops in NrdI is the one that interacts with the flavin ring of FMN, namely the 40s loop In the baNrdI structure, the loop extends from Thr42 to Pro49 (i.e eight residues) At the other extreme, in mycobacteria, the loop is up to 15 residues longer, containing a high proportion of Pro and Gly (Fig S1) The role of this highly variable length in a critical area of the structure is not clear Short- and long-chain flavodoxins differ in the presence or absence of a 20 residues loop that splits the fifth b-strand [23], although this is not the case in NrdI The 50s loop is slightly longer in long-chain Flds than in short-chain ones, and it has been proposed that the inserted loop stabilizes the longer 50s loop [42] However, an extended 40s loop in NrdI does not appear to be correlated with any other insertion It might be hypothesized that the extended loop contributes to increased affinity for NrdI’s interaction partner NrdF because, although the KD for the baNrdI ⁄ baNrdF complex is only 50 lm, the affinity of the E coli complex (with a loop longer by four residues) is so high that interactions are maintained during nickel–nitrilotriacetic acid affinity 4272 Cloning of the nrdI gene The B anthracis nrdI was amplified by PCR from strain Sterne 7700 pXO1) ⁄ pXO2) genomic DNA as described previously [19] using BanrdIup 5¢-ACATATGTTAGTTG CCTATGATTCTATG-3¢ and BanrdIlw 5¢-AAAGCTTAT TCAGTTCAATGTGTC-3¢, as a forward and reverse primers containing NdeI and HindIII restriction sites, respectively (underlined) The PCR product was cloned in the pGEM-T easy vector (Promega, Madison, WI, USA) After digestion with NdeI and HindIII, the nrdI fragment (380 bp) was ligated into pET22b generating plasmid pETS153 Expression and purification E coli Rosetta(DE3) cells (Novagen, Madison, WI, USA) containing pETS153 were grown in LB medium (Difco, Franklin Lakes, NJ, USA) at 37 °C with 100 lgỈmL)1 ampicillin, 17 lgỈmL)1 chloramphenicol and 60 lm FMN (Sigma, St Louis, MO, USA) until a A550 of 0.5 was reached, induced with mm isopropyl thio-b-d-galactoside for h, collected by centrifugation, and disrupted in an X-Press (BioX AB, Gothenburg, Sweden) in buffer 50 mm Tris-HCl (pH 7.6), 30 mm KCl and protease inhibitors FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS R Johansson et al (Roche Applied Science, Basel, Switzerland) All the protein purification steps were carried out at °C After highspeed centrifugation, the protein concentration was adjusted to 10 mgỈmL)1 and the supernatant solution was first precipitated with streptomycin sulfate (final concentration 1%) and, after a second centrifugation, with solid ammonium sulfate to 45% saturation After centrifugation, the precipitate was dissolved in buffer A (50 mm Tris-HCl, pH 7.6, and 30 mm KCl) and desalted by dialysis against L of buffer A for 16 h The dialyzed solution was diluted with buffer A to mgỈmL)1 protein concentration and loaded on HiLoad 16 ⁄ 10 Q-Sepharose High Performance column (GE Healthcare, Milwaukee, WI, USA) on a BioLogic DuoFlow System fast protein liquid chromatography instrument (Bio-Rad, Hercules, CA, USA) previously equilibrated with ten volumes of buffer A NrdI protein was eluted with a linear gradient of KCl (30–400 mm, mLỈmin)1) in buffer A Fractions containing the NrdI protein were pooled, concentrated using Centricon-10 (Millipore, Billerica, MA, USA) and loaded at 0.5 mLỈmin)1 on a 24-ml Superdex-75 column equilibrated and eluted with buffer 50 mm Tris-HCl (pH 7.6) and 200 mm KCl Each fraction was analyzed by PhastGel electrophoresis (GE Healthcare), and fractions with the highest purity (strong yellow colour) were pooled, concentrated using Centricon-10 (Millipore), and finally freed from KCl by washing with buffer A Crystallization and data collection The protein solution used for crystallization was at mgỈmL)1 in 50 mm Tris-HCl (pH 7.6) Screening for initial crystallization conditions was performed at the crystallization facility at MAX-lab The PACT Premier and JCSG+ screens (Molecular Dimensions Ltd, Newmarket, UK) were carried out at 20 °C in 100 + 100 nL drops in Greiner low-profile 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) A hit in condition 55 (E7) of the JCSG+ screen was refined using manual setups The crystal used for data collection on the oxidized form was grown from a drop consisting of lL of protein solution and lL of a reservoir solution consisting of 10% (v ⁄ v) 2-propanol, 0.2 m Zn acetate, 0.1 m Na cacodylate buffer (pH 6.5) Crystals appeared after one day and reached full size after approximately week The crystal used for data collection was approximately 0.2 · 0.1 · 0.1 mm in size Crystals were taken directly from the drop and flash-cooled in the gas stream from an Oxford Diffraction CryoJet (Oxford Diffraction Ltd, Oxford, UK) Data on the oxidized form ˚ were collected to 0.96 A resolution at station I911-3 of the MAX-II synchrotron (MAX-lab), using a 225 mm marMosaic CCD detector (Rayonix LLC, Evanston, IL, USA) ˚ The X-ray wavelength was 0.7300 A Data were collected, ˚ in two passes, to 1.9 and 0.96 A, respectively The crystal belonged to space group P212121 with cell dimensions as NrdI – an unusual flavodoxin Table Data and structure quality statistics Figures in parentheses refer to the highest resolution bin Oxidized a = 42.80, b = 45.62, c = 56.33 ˚ Unit cell dimensions (A) Data collection ˚ X-ray wavelength (A) ˚ Resolution range (A) Completeness (%) Rmerge (%) ⁄ Number of observations Number of unique reflections ˚ Wilson B-factor (A2) Refinement ˚ Resolution range (A) Rmodel (%) Rfree (%) Test set size, % (n) Number of protein residues Number of water molecules Other small molecules ˚ Mean isotropic B-factor (A2) Rmsd from ideal geometry ˚ Bond length (A) Bond angles Ramachandran plot quality Most favoured (%) Additional allowed (%) a From DANG restraints in Reduced a = 42.83, b = 45.26, c = 55.66 0.7300 23.9–0.96 (0.98–0.96) 97.3 (77.8) 3.9 (60.1) 17.1 (2.0) 283 726 65 490 11.1 0.9077 23.7–1.4 (1.44–1.4) 99.3 (98.7) 9.3 (74.4) 9.1 (1.5) 102 202 21 820 21.5 25–0.96 (1.0–0.96) 12.5 (25.9) 15.2 (–) 3.0 (1965) 118 185 · cacodylate, · Zn 13.4 (protein) 7.3 (FMN) 34.2 (water) 26.7–1.4 (1.46–1.40) 14.7 (23.8) 19.2 (32.5) 5.0 (1106) 118 120 · cacodylate, · Zn 14.3 (protein), 10.5 (FMN) 39.2 (water) 0.016 ˚ 0.035 Aa 0.024 1.99° 99.2 0.8 99.2 0.8 SHELXL shown in Table For determination of the structure of reduced NrdI, a crystal was soaked in a solution consisting of 500 mm sodium dithionite in crystallization mother liquor for 10 before flash cooling During this time, the crystal colour changed from yellow to dark blue Data were collected at station I911-5 of the MAX-II synchrotron, with ˚ an X-ray wavelength of 0.9789 A, in two passes, to 1.8 and ˚ , respectively Diffraction data were integrated using 1.4 A xds and scaled using xscale [44] Data were further processed using software from the ccp4 suite [45] The crystals contain one NrdI molecule in the asymmetric unit, resulting ˚ in a Matthews volume of 2.05 A3ỈDa)1 and a solvent content of 40.0% Structure solution and refinement The structure of oxidized baNrdI was solved by molecular replacement (MR) using the unpublished coordinates of FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS 4273 NrdI – an unusual flavodoxin R Johansson et al NrdI from Bacillus subtilis (PDB code: 1RLJ), which has 48% sequence identity to baNrdI The automated MR pipeline mrbump [46] was used The best search model was generated by truncation of nonconserved side chains using molrep from the ccp4 suite and the solution was found using molrep as the MR search engine The MR solution had an R-factor of 36.0% and a free R-factor of 37.3% (calculated using 3% of the data) The model was rebuilt by iterated rounds of model building in coot [47] and refinement in refmac5 [48] The high-resolution limit for refine˚ ˚ ment was increased gradually from 2.0 A to 1.1 A ˚ Anisotropic B-factors were introduced at 1.5 A After convergence of refinement in refmac5, Rmodel and Rfree were 15.6% and 18.1%, respectively At this point, the refinement software was switched to shelxl [49] Restraints for FMN were generated from coordinates in the HIC-UP database [50] using the prodrg server [51] To ensure convergence, ˚ the resolution was reduced to 1.8 A and gradually increased ˚ to 0.96 A Riding hydrogen atoms were used throughout ˚ Anisotropic B-factors were introduced at 1.6 A resolution After shelxl refinement, Rmodel and Rfree were 13.5% and 16.3%, respectively Water molecules were introduced in peaks over 4.0 r in the difference map fulfilling reasonable distance and hydrogen bonding criteria to protein residues or other water molecules Refined water molecules were removed if they had excessively high B-factors or electron density in 2Fo ) Fc maps under 1.0 r The structure of reduced baNrdI was solved by direct refinement of the oxidized structure against the dataset from a reduced crystal After the first round of refinement, strong difference electron density was observed, indicating a flip in the peptide bond between residues 44 and 45 The solvent structure was modelled according to the same criteria as for the oxidized protein and the coordinates were refined using refmac5 [48] and phenix.refine [52] Electrostatic potential calculations Electrostatic potentials were calculated using the apbs plugin [53] to pymol (http://www.pymol.org) using default parameters throughout Sequence analysis A set of 199 unique NrdI sequences was extracted from the RNRdb database: sequences annotated as containing only an NrdI fragment were immediately discarded Sequences from different strains of the same organism were then removed to decrease redundancy In cases where different strains contained either one or two NrdI sequences, the strain containing two sequences was retained The remaining sequences were aligned using clc sequence viewer 6.3 ˚ (CLC Bio, Arhus, Denmark; http://www.clcbio.com) Four sequences that were not marked as fragments in the database, but which were evidently too short because they 4274 lacked the first a-helix and b-strand, were then removed Isoelectric points, sequence lengths and amino acid compositions were calculated and tabulated using clc sequence viewer 6.3 and overall statistics calculated using Microsoft Excel (Microsoft Corp., Redmond, CA, USA) For comparison, an analysis of 38 flavodoxin sequences extracted from the UniProt100 database (http://www.uniprot.org) was also carried out Phylogenetic reconstruction From the 277 unique NrdI sequences in the RNRdb [7], 91 representative sequences were chosen and aligned using probcons, version 1.10 [54] The sequences were chosen to represent the full diversity of NrdI sequences except for highly divergent sequences A maximum likelihood tree was estimated from 100 well-aligned positions using phyml, version 3.0, the LG substitution model and four gamma categories [55] Branch confidence was calculated using the SH-like algorithm [56] Estimation of isoelectric points pI values of protein sequences were estimated using the pI ⁄ Mw tool at the expasy web server (http://www.expasy ch/tools/pi_tool.html) Acknowledgements This work was supported by grants from the Swedish Research Council to B.M.S and D.L E.T was supported by grants from the Spanish Ministerio de ´ Ciencia e Innovacion (PI081062), the CONSOLIDER (CSD2008-00013) and ERANET Pathogenomics.We ˚ wish to thank Maria Hakansson for help at the MAXlab crystallization facility and the staff at beamline I911 at MAX-lab for assistance with data collection We thank Ilya Borovok for stimulating discussions References ˚ Eklund H, Uhlin U, Farnegardh M, Logan DT & ¨ Nordlund P (2001) Structure and function of the radical enzyme ribonucleotide reductase Prog Biophys Mol Biol 77, 177–268 Nordlund P & Reichard P (2006) Ribonucleotide reductases Annu Rev Biochem 75, 681–706 Torrents E, Sahlin M & Sjoberg BM (2008) The ribonuă ceotide reductase family: genetics and genomics In Ribonucleotide Reductase (Andersson KK ed), pp 17–77 Nova Science Publishers, New York Jordan A, Pontis E, Aslund F, Hellman U, Gibert I & Reichard P (1996) The ribonucleotide reductase system of Lactococcus lactis Characterization of an NrdEF FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS R Johansson et al 10 11 12 13 14 15 16 17 enzyme and a new electron transport protein J Biol Chem 271, 8779–8785 Eliasson R, Pontis E, Jordan A & Reichard P (1996) Allosteric regulation of the third ribonucleotide reductase (NrdEF enzyme) from enterobacteriaceae J Biol Chem 271, 26582–26587 Eriksson M, Jordan A & Eklund H (1998) Structure of Salmonella typhimurium nrdF ribonucleotide reductase in its oxidized and reduced forms Biochemistry 37, 13359–13369 Lundin D, Torrents E, Poole AM & Sjoberg BM (2009) RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank BMC Genomics 10, 589 Willing A, Follmann H & Auling G (1988) Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme Eur J Biochem 170, 603–611 Fieschi F, Torrents E, Toulokhonova L, Jordan A, Hellman U, Barbe J, Gibert I, Karlsson M & Sjoberg ă BM (1998) The manganese-containing ribonucleotide reductase of Corynebacterium ammoniagenes is a class Ib enzyme J Biol Chem 273, 4329–4337 Huque Y, Fieschi F, Torrents E, Gibert I, Eliasson R, Reichard P, Sahlin M & Sjoberg BM (2000) The active ă form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein J Biol Chem 275, 25365–25371 Cotruvo JA Jr & Stubbe J (2008) NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase Proc Natl Acad Sci USA 105, 14383–14388 Cotruvo JA Jr & Stubbe J (2010) An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase Biochemistry 49, 1297– 1309 Jordan A, Aragall E, Gibert I & Barbe J (1996) Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria Mol Microbiol 19, 777–790 ˚ Jordan A, Aslund F, Pontis E, Reichard P & Holmgren A (1997) Characterization of Escherichia coli NrdH A glutaredoxin-like protein with a thioredoxin-like activity profile J Biol Chem 272, 18044–18050 Roca I, Torrents E, Sahlin M, Gibert I & Sjoberg BM (2008) NrdI essentiality for class Ib ribonucleotide reduction in Streptococcus pyogenes J Bacteriol 190, 4849–4858 Stubbe J & van Der Donk WA (1998) Protein radicals in enzyme catalysis Chem Rev 98, 705–762 Draper RD & Ingraham LL (1968) A potentiometric study of the flavin semiquinone equilibrium Arch Biochem Biophys 125, 802–808 NrdI – an unusual flavodoxin 18 Hoover DM, Drennan CL, Metzger AL, Osborne C, Weber CH, Pattridge KA & Ludwig ML (1999) Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans Structural determinants of the redox potentials J Mol Biol 294, 725–743 19 Torrents E, Sahlin M, Biglino D, Graslund A & ¨ Sjoberg BM (2005) Efficient growth inhibition of ¨ Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical Proc Natl Acad Sci USA 102, 17946–17951 20 Røhr AK, Hersleth HP & Andersson KK (2010) Tracking flavin conformations in protein crystal structures with Raman spectroscopy and QM ⁄ MM calculations Angew Chem Int Ed Engl 49, 2324–2327 21 Holm L & Sander C (1995) Dali: a network tool for protein structure comparison Trends Biochem Sci 20, 478–480 22 Guelker M, Stagg L, Wittung-Stafshede P & Shamoo Y (2009) Pseudosymmetry, high copy number and twinning complicate the structure determination of Desulfovibrio desulfuricans (ATCC 29577) flavodoxin Acta Crystallogr D Biol Crystallogr 65, 523–534 23 Sancho J (2006) Flavodoxins: sequence, folding, binding, function and beyond Cell Mol Life Sci 63, 855– 864 24 Kleywegt GJ, Harris MR, Zou JY, Taylor TC, Wahlby A & Jones TA (2004) The Uppsala Electron-Density Server Acta Crystallogr D Biol Crystallogr 60, 2240– 2249 25 Smith WW, Burnett RM, Darling GD & Ludwig ML (1977) Structure of the semiquinone form of flavodoxin from Clostridium MP Extension of 1.8 A resolution and some comparisons with the oxidized state J Mol Biol 117, 195–225 26 O’Farrell PA, Walsh MA, McCarthy AA, Higgins TM, Voordouw G & Mayhew SG (1998) Modulation of the redox potentials of FMN in Desulfovibrio vulgaris flavodoxin: thermodynamic properties and crystal structures of glycine-61 mutants Biochemistry 37, 8405–8416 27 McCarthy AA, Walsh MA, Verma CS, O’Connell DP, Reinhold M, Yalloway GN, D’Arcy D, Higgins TM, Voordouw G & Mayhew SG (2002) Crystallographic investigation of the role of aspartate 95 in the modulation of the redox potentials of Desulfovibrio vulgaris flavodoxin Biochemistry 41, 10950–10962 28 Paithankar KS & Garman EF (2010) Know your dose: RADDOSE Acta Crystallogr D Biol Crystallogr 66, 381–388 29 Bollen YJ, Nabuurs SM, van Berkel WJ & van Mierlo CP (2005) Last in, first out: the role of cofactor binding in flavodoxin folding J Biol Chem 280, 7836–7844 30 Wassink JH & Mayhew SG (1975) Fluorescence titration with apoflavodoxin: a sensitive assay for riboflavin 5¢-phosphate and flavin adenine dinucleotide in mixtures Anal Biochem 68, 609–616 FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS 4275 NrdI – an unusual flavodoxin R Johansson et al 31 Genzor CG, Perales-Alcon A, Sancho J & Romero A (1996) Closure of a tyrosine ⁄ tryptophan aromatic gate leads to a compact fold in apo flavodoxin Nat Struct Biol 3, 329–332 32 Martinez-Julvez M, Cremades N, Bueno M, Perez-Dorado I, Maya C, Cuesta-Lopez S, Prada D, Falo F, Hermoso JA & Sancho J (2007) Common conformational changes in flavodoxins induced by FMN and anion binding: the structure of Helicobacter pylori apoflavodoxin Proteins 69, 581–594 33 Watt W, Tulinsky A, Swenson RP & Watenpaugh KD (1991) Comparison of the crystal structures of a flavodoxin in its three oxidation states at cryogenic temperatures J Mol Biol 218, 195–208 34 Romero A, Caldeira J, Legall J, Moura I, Moura JJ & Romao MJ (1996) Crystal structure of flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in two oxidation states Eur J Biochem 239, 190–196 35 Ludwig ML, Pattridge KA, Metzger AL, Dixon MM, Eren M, Feng Y & Swenson RP (1997) Control of oxidation-reduction potentials in flavodoxin from Clostridium beijerinckii: the role of conformation changes Biochemistry 36, 1259–1280 36 Ishikita H (2007) Influence of the protein environment on the redox potentials of flavodoxins from Clostridium beijerinckii J Biol Chem 282, 25240–25246 37 Ishikita H (2008) Redox potential difference between Desulfovibrio vulgaris and Clostridium beijerinckii flavodoxins Biochemistry 47, 4394–4402 38 Zhou Z & Swenson RP (1995) Electrostatic effects of surface acidic amino acid residues on the oxidationreduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough) Biochemistry 34, 3183– 3192 39 Goni G, Herguedas B, Hervas M, Peregrina JR, De la Rosa MA, Gomez-Moreno C, Navarro JA, Hermoso JA, Martinez-Julvez M & Medina M (2009) Flavodoxin: a compromise between efficiency and versatility in the electron transfer from photosystem I to ferredoxinNADP(+) reductase Biochim Biophys Acta 1787, 144– 154 40 Massey V (1994) Activation of molecular oxygen by flavins and flavoproteins J Biol Chem 269, 22459– 22462 ´ 41 Lindqvist Y & Branden CI (1989) The active site of ă spinach glycolate oxidase J Biol Chem 264, 3624–3628 ´ 42 Lopez-Llano J, Maldonado S, Bueno M, Lostao A, ´ ´ Angeles-Jimenez M, Lillo MP & Sancho J (2004) The long and short flavodoxins: I The role of the differentiating loop in apoflavodoxin structure and FMN binding J Biol Chem 279, 47177–47183 43 Boal AK, Cotruvo JA Jr, Stubbe J & Rosenzweig AC(2010) Structural Basis for Activation of Class Ib Ribonucleotide Reductase Science doi:10.1126/ science.1190187 4276 44 Kabsch W (2010) XDS Acta Crystallogr D Biol Crystallogr 66, 125–132 45 Collaborative Computational Project n (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D Biol Crystallogr 50, 760–763 46 Keegan RM & Winn MD (2007) Automated searchmodel discovery and preparation for structure solution by molecular replacement Acta Crystallogr D Biol Crystallogr 63, 447–457 47 Emsley P, Lohkamp B, Scott WG & Cowtan K (2010) Features and development of Coot Acta Crystallogr 66, 486–501 48 Murshudov GN, Vagin AA & Dodson EE (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr D Biol Crystallogr 53, 240–255 49 Sheldrick GM (2008) A short history of SHELX Acta Crystallogr A 64, 112–122 50 Kleywegt GJ & Jones A (1998) Databases in protein crystallography Acta Crystallogr D Biol Crystallogr 54, 1119–1131 51 van Aalten DM, Bywater R, Findlay JB, Hendlich M, Hooft RW & Vriend G (1996) PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules J Comput Aided Mol Des 10, 255–262 52 Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution Acta Crystallogr D Biol Crystallogr 66, 213–221 53 Baker NA, Sept D, Joseph S, Holst MJ & McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome Proc Natl Acad Sci USA 98, 10037–10041 54 Do CB, Mahabhashyam MS, Brudno M & Batzoglou S (2005) ProbCons: Probabilistic consistency-based multiple sequence alignment Genome Res 15, 330–340 55 Guindon S & Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood Syst Biol 52, 696–704 56 Anisimova M & Gascuel O (2006) Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative Syst Biol 55, 539–552 Supporting information The following supplementary material is available: Fig S1 Multiple sequence alignment of 199 NrdI sequences extracted from the RNRdb Fig S2 Electron density revealing a possible metal ion in the crystal structure of the reduced form of B cer˚ eus NrdI at 1.15 A resolution FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS R Johansson et al Fig S3 Maximum likelihood phylogeny of a representative choice of 91 NrdI proteins Table S1 Lengths of secondary structure elements in baNrdI compared to the closest structural neighbour, the flavodoxin from Anacystis nidulans (PDB code: 3F6R) This supplementary material can be found in the online version of this article NrdI – an unusual flavodoxin Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 4265–4277 ª 2010 The Authors Journal compilation ª 2010 FEBS 4277 ... 50s and 90s loops [23] We refer to these as the 40s and 70s loops, respectively, in baNrdI The flavin moiety is sandwiched between Trp74 on one face and the side chain of Thr42 and the main chain... preceding P-loop The flavin moiety of FMN binds in a pocket formed by loops at the C-terminal ends of b-strands and (loops 4 2–4 9 and 7 1–7 9, respectively), known in Fld as the W and Y loops, or the. .. details of FMN binding are very similar to baNrdI; however, note the preponderance of acidic residues in the vicinity of the FMN binding site Prepared using PYMOL 4268 N-terminus of a1 and the preceding