Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification.

EDITOR’S CHOICE

Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongatordependent tRNA modification raphin1 Olga Kolaj-Robin1, Alastair G. McEwen2, Jean Cavarelli2 and Bertrand Se e La Ligue, Institut de Ge ne tique et de Biologie Mole culaire et Cellulaire, Centre National de Recherche Scientifique UMR 1 Equipe Labellise et de Recherche Me dicale U964/Universite de Strasbourg, Illkirch, France 7104/Institut National de Sante ne tique et de Biologie Mole culaire et Cellulaire, Centre National de Recherche Scientifique UMR 7104/Institut National de 2 Institut de Ge et de Recherche Me dicale U964/Universite de Strasbourg, Illkirch, France Sante

Keywords electron transfer; elongator; Kti11/Kti13; Saccharomyces cerevisiae; tRNA modification Correspondence raphin, Institut de Ge ne tique et de B. Se culaire et Cellulaire, 1 rue Biologie Mole Laurent Fries, 67404 Illkirch, France Fax: +33 3 88 65 32 01 Tel: +33 3 88 65 33 56 E-mail: [email protected] (Received 1 December 2014, revised 12 January 2015, accepted 14 January 2015) doi:10.1111/febs.13199

Modification of wobble uridines of many eukaryotic tRNAs requires the Elongator complex, a highly conserved six-subunit eukaryotic protein assembly, as well as the Killer toxin-insensitive (Kti) proteins 11–14. Kti11 was additionally shown to be implicated in the biosynthesis of diphthamide, a post-translationally modified histidine of translation elongation factor 2. Recent data indicate that iron-bearing Kti11 functions as an electron donor to the [4Fe–4S] cluster of radical S-Adenosylmethionine enzymes, triggering the subsequent radical reaction. We show here that recombinant yeast Kti11 forms a stable 1 : 1 complex with Kti13. To obtain insights into the function of this heterodimer, the Kti11/Kti13 complex was purified to resolution. homogeneity, crystallized, and its structure determined at 1.45 A The importance of several residues mediating complex formation was confirmed by mutagenesis. Kti13 adopts a fold characteristic of RCC1-like proteins. The seven-bladed b-propeller consists of a unique mixture of fourand three-stranded blades. In the complex, Kti13 orients Kti11 and restricts access to its electron-carrying iron atom, constraining the electron transfer capacity of Kti11. Based on these findings, we propose a role for Kti13, and discuss the possible functional implications of complex formation. Database Structural data have been submitted to the Protein Data Bank under accession number 4X33.

Introduction Kti11 (Killer toxin-insensitive 11) from Saccharomyces cerevisiae is a small, highly conserved protein, currently described as a CSL zinc-finger family member (named after the final conserved cysteine of the zinc finger and the next two residues). Homologous proteins are found in most eukaryotes. Together with genes encoding Elongator complex subunits (Elp1-6),

Kti11 was identified as one of the factors maintaining the sensitivity of S. cerevisiae to Kluveromyces lactis zymocin [1,2]. The latter has been shown in vitro to target tRNAs carrying 5-methoxycarbonylmethyl-2thiouridine nucleosides at the wobble position [3,4]. The Elongator complex is a highly conserved sixsubunit eukaryotic protein assembly that has been

Abbreviations DESR1, diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) sensitivity required gene 1; eEF2, eukaryotic translation elongation factor 2; GEF, guanine exchange factor; GIP, GEF interacting protein 1; ITC, isothermal titration calorimetry; Kti, Killer toxin-insensitive; RLD, RCC1like domain; RPGR, retinitis pigmentosa GTPase regulator; SAM, S-Adenosylmethionine.

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Analysis of the Kti11/Kti13 complex

implicated in several cellular functions, including generation of modified 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) wobble uridines across species from yeast to mice [5–8]. The co-purification of Kti11 with the Elp1–3 sub-complex [9], together with its ability to co-precipitate Elp2 and Elp5 [10], confirmed its strong functional relationship with Elongator. Yeast Kti11 was independently found to be identical to Dph3, and to interact with diphthamide synthesis factors Dph1 and Dph2. Together, Kti11/Dph3 and Dph1–2 are required for the first step of biosynthesis of diphthamide [9,11–13], a post-translationally modified histidine residue in eukaryotic translation elongation factor 2 (eEF2). This modification creates a target for diphtheria toxin, which inactivates eEF2 by ADP-ribosylation of the diphtamide residue, eventually causing cell death [14,15]. Accordingly, kti11D mutants resist diphtheria toxin, protect against ADP-ribosylation of eEF2 by the toxin, and induce resistance against sordarin, an eEF2-poisoning antifungal agent [10]. Among many other versatile partners such as ribosomal proteins or eEF2, yeast Kti11 was also shown to interact with Kti13, a factor also known as Ats1 [16]. Sequence comparisons indicate that Kti13 contains a RCC1 domain and thus is a Ran GEF-like protein (Ran guanine exchange factor-like protein). Deletion of kti13 in yeast protects against zymocin [1,2], and modification of wobble uridine nucleosides in this mutant is significantly decreased [5,17], suggesting an important but not crucial functional relationship between Kti13, Kti11 and Elongator. Yeast Kti13 was also reported to interact with cell polarity factor Nap1 [18,19]. In humans, a putative guanine nucleotide exchange factor protein, DelGEF, that exhibits significant homology to Kti13, is located in a deafness locus, and was found to be critical for two forms of hereditary deafness [20]. DelGEF was reported to interact with Sec5, a subunit of a multi-protein secretory complex [21]. Together with its partner DelGIP1/ DESR1, the Kti11 human homologue, DelGEF was reported to affect proteoglycan secretion by an unknown mechanism [22]. An indirect link of yeast Kti11, Kti13 and Elongator to the secretion process has also been reported, as various yeast secretory mutations are suppressible by kti and elp deletions [19]. However, this effect was reversed by increased levels of Elongator-dependent tRNAs [23], suggesting that altered translation was primarily responsible for defective secretion. The high conservation and wide distribution of Kti11 in eukaryotes suggest that it has an important cellular function. Consistently, loss of Kti11 was shown to cause embryonic lethality in mice [24]. 820

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Although initially described as a zinc-binding protein [25], Kti11 was shown to be also capable of binding iron, and confirmed to be a redox-active protein [26]. In line with these observations, the role of Kti11 in the synthesis of diphthamide was recently shown to donate electrons to the [4Fe–4S] cluster-containing Dph2 present in Dph1–Dph2 dimers [12]. In turn, this triggers synthesis of diphthamide using an unusual S-Adenosylmethionine (SAM) radical reaction [27]. Kti11 may also pass electrons to Elp3 of the Elongator complex. Indeed, the presence of a [4Fe–4S] cluster in eukaryotic Elp3 has been proposed based on sequence comparisons [28]. This possibility is further supported by the identification of an [4Fe–4S] cluster in archaeal Elp3 homolgues that were also shown to bind SAM [29]. The recent observation of the modification of tRNA wobble uridine by a [4Fe–4S]-containing archaeal homologue of Elp3 in a process involving a SAMmediated radical mechanism further demonstrates the biochemical relationship between the prokaryotic and eukaryotic systems [30]. Similarities of the chemical mechanisms implicated in diphtamide biosynthesis and wobble uridine modification in archaea strengthen the hypothesis that Kti11 acts in eukaryotes as an electron donor to the Elp3 subunits of Elongator, although this awaits final confirmation. However, the role of Kti13 remains elusive. Its involvement in Elongator-related function, although not crucial, is well demonstrated. So far, however, there is no indication that Kti13 is engaged in diphthamide synthesis, while Kti11 is required for both those processes. To obtain insight into the role of Kti13 and its association with Kti11, we performed a functional and structural analysis of this complex. Our data suggest a role for Kti13 in modulating the ability of Kti11 to transfer electrons.

Results Kti11 and Kti13 form a stable complex Using tagged proteins expressed in yeast, Kti11 was previously shown to co-immunoprecipitate with Kti13 [19]. After independently confirming this observation (data not shown), we investigated whether Kti11 and Kti13 interact directly. For this purpose, we constructed two bi-cistronic expression vectors: one encoding full-length Kti11 and Kti13, while the second encoded a C-terminally truncated Kti11 (residues 1– 57) with full-length Kti13. The solution structure of Kti11 indicates that the 25 C-terminal amino acids of Kti11 form an a-helix that protrudes from the otherwise globular structure [25], while co-immunoprecipitation experiments showed that the C-terminal region of FEBS Journal 282 (2015) 819–833 ª 2015 FEBS



Kti11 is not required for pull-down of Kti13 [10]. Proteins recombinantly expressed in Escherichia coli copurified upon affinity purification using the His6 tag fused to Kti11, indicating that they form a stable heterodimeric complex (Fig. 1A, and data not shown). Because subsequent crystallization trials did not yield protein crystals with the full-length proteins, we concentrated our efforts on the Kti11(1–57)-His6/Kti13 complex. Size-exclusion chromatography showed two main peaks eluting at approximately 14 and 16.5 mL, corresponding to the Kti11/Kti13 complex and to free Kti11, respectively. In addition to 280 nm, elution of the latter was followed at 480 nm due to the presence of an oxidized iron. The complex was highly homogenous, as the size distribution histogram of the purified Kti11(1–57)-His6/Kti13 complex determined using dynamic light scattering demonstrated the presence of a mono-modal mono-disperse protein (%Pd of approximately 11%) protein with a hydrodynamic radius of approximately 2.96 nm (Fig. 1B). The estimated molecular mass of the complex by dynamic light scattering was 43 kDa, assuming a globular structure, close to the calculated theoretical value of 43 915 Da. Complex formation was further studied by isothermal titration calorimetry (ITC) by mixing recombinant, independently purified His6-tagged Kti11 and His6-tagged Kti13. This analysis revealed a stoichiometric coefficient of 0.76 0.049 and a dissociation constant kD of 0.25 0.11 lM, supporting the biological relevance of the complex (Fig. 1C). The positive enthalpy of the process implies that hydrophobic effects play a substantial role in complex formation. The additional small peak observed after the A Fig. 1. Kti11 and Kti13 form a stable complex. (A) Size-exclusion chromatography purification profile and SDS/PAGE analysis of the Kti11(1–57)His6Kti13 complex. Lanes labelled 1 and 2 on the SDS/PAGE gel represent the fraction corresponding to the top of the peaks on the chromatogram. (B) Size distribution histogram of purified Kti11/Kti13 sample subjected to crystallization trials. (C) ITC analysis of His6-tagged Kti11 and His6tagged Kti13 complex formation. The upper and the lower plots show the raw heats of binding and the integrated heats of binding, excluding dilution effects, respectively. Values in the box are means obtained from six independent ITC runs with two batches of His-tagged Kti11 protein.


main titration event remained beyond our interpretation. However, it is noteworthy that only a one-site model but not a two-site model could be fitted to these data. Altogether, these data indicate that Kti11 and Kti13 interact directly with good affinity, and form a stable heterodimeric complex in the absence of any other yeast proteins. Crystal structure of Kti11/Kti13 complex The complex of the truncated Kti11 (amino acids 1– 57) together with full-length Kti13 crystallized in the C2221 orthorhombic space group, with one copy of resoluthe complex per asymmetric unit. The 1.45 A tion structure (Fig. 2A) contains residues 2–57 of Kti11 with an additional four C-terminal histidines, as well as all residues of Kti13 except residues 32–34 and 281–284, which form non-conserved flexible loops. Kti11 binds one iron atom coordinated by four conserved cysteine residues (C25, C27, C47 and C50) (Fig. 2B) as previously described [25,26], forming an Fe(Cys)4 centre with distorted tetrahedral geometry. The presence of only iron, and not zinc, ions in Kti11/Kti13 crystals was demonstrated by collecting an X-ray fluorescence spectrum for the crystal (data not shown) and two fluorescence scans around the and absorption edges of iron (approximately 1.73 A) zinc (approximately 1.28 A) prior to data collection. The presence of iron was later confirmed by anomalous difference Fourier maps using phases from the final model. The Kti11 crystal structure is similar to and may be superposed on the previously reported C

B

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A

B

Fig. 2. Structure of S. cerevisiae Kti11(1– 57)His6/Kti13 complex. (A) Overall structure of the yeast Kti11(1–57)His6/ Kti13 complex. (B) Enlargement of the area indicated in (A). The iron-coordinating cysteine residues of Kti11, H297 of Kti13 that points directly towards the iron atom, and the Kti13 tunnel-closing residue C326 are shown as sticks and labelled.

NMR structure of the protein (PDB ID 1YOP [25]), as well as with two known structures of Kti11 homologues: human and mouse DelGIP/DESR1 (PDB IDs 2JR7 and 1WGE, respectively), with RMSD values in the range 1.00–1.51 over 57 Ca atoms. The larger component of the complex, Kti13, comprises one 310 helix and 26 b-strands. Kti13 contains one canonical RCC1 repeat according to the Pfam database (http:// pfam.xfam.org/), adopting the seven-bladed b-propeller fold that is characteristic of proteins belonging to this superfamily (Fig. 2A) [31]. The structure also shows the characteristic 2+2 arrangement of the first/ last b-sheet with two strands from the N-terminal half repeat and two from the C-terminal half repeat, similar to other RCC1-like proteins. Interestingly, however, the structure of Kti13 exhibits significant deviations from the canonical fold of eukaryotic RCC1-like proteins in which the blades of the propeller are similar to each other and each consists of four antiparallel b-strands. Instead, two blades of yeast Kti13, namely five and six, consist of only three antiparallel b-strands, while the loop that connects them

A

additionally contains a 310 helix in place of the fourth strand in blade 5 (Fig. 3). These resemble blades found in bacterial b-lactamase inhibitor protein II, a protein that exhibits a fold that is highly similar to that of eukaryotic RCC1-like proteins except that each bade is composed of three antiparallel b-strands followed by a helix-containing loop [32]. Superimposition of yeast Kti13 with bacterial b-lactamase inhibitor protein II (Fig. 3A) and with human retinitis pigmentosa GTPase regulator (RPGR; Fig. 3B) yields RMSD values of 2.09 and 2.24 over 223 and 296 Ca atoms, respectively. Additionally, superposition of its individual blades clearly illustrates these differences (data not shown). The yeast Kti13 propeller therefore represents an intermediate form of seven-bladed propellers between bacteria and higher eukaryotes. The inner strands of each blade surround a central tunnel filled with water molecules. The volume and the effec3 tive radius of the tunnel were calculated as 1243 A and 5.21 A, respectively, using the 3V web server [33]. It is closed by a relatively well-conserved Cys residue (C326) at the end where Kti13 interacts with Kti11

B

Fig. 3. Yeast Kti13 combines structural features of b-propellers from bacterial and higher eukaryotic species. Superposition of S. cereviasiae Kti13 (green) with bacterial b-lactamase inhibitor protein II (PDB ID 1JTD) (blue) (A) and with retinitis pigmentosa GTPase regulator (PDB ID 4JHN) (pink) (B).The blades of the propellers are numbered from 1 to 7.

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complex was observed for W96A/K157D and W96A/ W229C but not the W294E/H297A double mutant. Size-exclusion chromatography of the latter showed only partial dissociation of the complex, suggesting that the conserved residues, especially the iron-facing H297, may have additional roles besides maintaining the association of the two proteins. Overall, this mutant analysis confirms the structural data and the importance of conserved hydrophobic contacts, particularly mediated by tryptophan residues, for the association of Kti13 with Kti11. Kti11/Kti13 and electron transfer Iron-containing Kti11 exhibits spectroscopic features characteristic of rubredoxins, a family of small electron carriers. Accordingly, it was shown to be able to accept electrons from the general rubredoxin reductase NorW from E. coli [26]. It is also capable of further transferring them to horse heart cytochrome c, an electron acceptor that is widely used in in vitro redox analysis [26]. Given the recent report that Kti11 serves as an electron donor to Dph1-2 and most likely to the Elongator complex, and our structural data indicating that access to the iron atom is sterically restricted in the Kti11–Kti13 complex (Fig. 2), we decided to anaA

lyse the influence of Kti13 on the electron transfer abilities of Kti11. In the absence of Kti11, a basal anaerobic reduction of cytochrome c in the presence of NorW (0.5 lM) and NADH (115 lM) was observed (Fig. 8A). This is demonstrated by increased absorbance at 550 nm, a wavelength that corresponds to maximum absorption of reduced cytochrome c (Fig. 8A; inset). Upon addition of Kti11 (2 lM), the rate of the reaction increases drastically. Addition of Kti13 in the molar ratio 1 : 3 significantly reduced the rate of cytochrome c reduction, which reached the level of basal reaction when both proteins are present in equimolar amounts. The speed of the reaction remained at the basal level upon addition of a twofold molar excess of Kti13 (Fig. 8A). The spectrum recorded in the 650–400 nm range approximately 30 min after the end of each reaction was identical to that obtained by reduction of cytochrome c with dithionite, demonstrating that re-oxidation of the cytochrome c did not occur during the experiments, and thus did not account for the effects mediated by Kti13 (data not shown). Interestingly, upon complex dissociation by treatment at 50°C for 2 h, the rate of cytochrome c reduction was restored to the level observed when Kti11 was present alone (data not shown). Moreover, Kti11 may be efficiently reduced using B

C

Fig. 8. Redox activity of Kti11 alone and in complex with Kti13. (A) Reduction of horse heart cytochrome c by His6-tageed Kti11 alone (black) and in the presence of His6-tagged Kti13 in Kti11:Kti13 molar ratios of 3 : 1 (green), 1 : 1 (red) and 1 : 2 (blue). The background reaction in the absence of Kti11 and Kti13 is shown in grey. Inset: air-oxidized (red) and dithionite-reduced (black) spectra for horse heart cytochrome c. (B) Reduction of the His6-tagged Kti11 (bold line) and Kti11(1–57)-His6/Kti13 complex (thin line) by NorW. The addition of NADH for both reactions and dithionite (1 mM) for analysis of reduction of Kti11 in complex with Kti13 are indicated by black and red arrows, respectively. (C) Reduction of horse heart cytochrome c by previously reduced His6-tagged Kti11 alone (black) and in the presence of an equimolar amount of His6-tagged Kti13 (red). The background reaction in the absence of both Kti11 and Kti13 is shown in grey.


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dithionite even if it is in complex with Kti13 (Fig. 8B). This demonstrates that, upon complexing with Kti13, Kti11 is not irreversibly inactivated but rather sterically inhibited. These results demonstrate a clear influence of Kti13 on Kti11’s electron transfer ability, as the latter cannot participate in the electron transfer chain when Kti13 is present. To identify the step affected, we analysed the consequences of complexing of Kti13 with Kti11 on reduction of the latter by NorW in the presence of NADH. This was monitored by following absorbance at 488 nm, a wavelength at which oxidized but not reduced Kti11 exhibits maximum absorption [26]. The results shown in Fig. 8B demonstrate that electron transfer between NorW and Kti11 was no longer observed in the presence of Kti13. As NorW is a non-specific electron donor for Kti11, it is possible that the consequences of complexing of the latter with Kti13 do not have as drastic effect on the electron transfer between Kti11 and its endogenous reductase in vivo. Nevertheless, the results clearly show that Kti13 restricts access to Kti11. In order to verify whether complexing of Kti11 with Kti13 also affects the electron transfer from Kti11 to cytochrome c, prior reduction of Kti11 in the reaction was required. Therefore, Kti11 was first anaerobically mixed with NADH and NorW, after which Kti13 was added, followed by addition of cytochrome c. Such an experimental set-up ensures formation of the Kti11/ Kti13 complex after reduction of Kti11 by NorW. As shown in Fig. 8C, the presence of Kti13 significantly decreased the speed of cytochrome c reduction by Kti11; however, this remains well above the background reaction. This indicates that electron transfer to cytochrome c is decreased when Kti13 is present. In summary, our data clearly demonstrate that the presence of Kti13 restricts access to the iron atom of Kti11, thus affecting the specificity of the electron transfer reactions.

Discussion The small, widely distributed and conserved eukaryotic protein Kti11 has been shown to functionally interact with two protein complexes, Elongator and Dph1–2, which are involved in modification of tRNA wobble uridines and diphthamide biosynthesis, respectively. Deletion of the gene encoding Kti11 results in many cellular defects and causes embryonic lethality in mice, emphasizing its importance in the cell. Recent findings support the idea that Kti11 functions by delivering electrons to the [4Fe–4S] clusters of its partner complexes, triggering radical reactions [12,30]. Kti11 has been shown to co-precipitate with Kti13, and the latter 826


has been reported to be required for efficient modification of tRNA wobble uridines but not for diphthamide biosynthesis. However, the function of Kti13 has not been characterized in detail. We have shown that Kti13 forms a stable heterodimeric complex with Kti11, and determined the crystal structure of this resolution. In the crystallized heteassembly at 1.45 A rodimer, Kti11 contains iron. The iron atom is bound by four cysteines of Kti11 that form part of the and sequence Cys-Pro-Cys-X19-Cys-Pro-Ser-Cys, shows distorted tetrahedral geometry. The mean Fe–S (2.32 A for C25, 2.36 A for C27, distance is 2.317 A 2.34 A for C47, and 2.25 A for C50). Three of these values are in the expected range for the Fe2+ oxidation state [35,36], but it is not possible to unambiguously distinguish between the two iron oxidation states. Further analyses are required to determine the iron oxidation state, especially as reduction by photoelectrons may have occurred during data collection. Kti13 adopts a seven-bladed propeller fold characteristic of RCC1-like domains (RLDs). Analyses of several RLDs demonstrated that they perform many different functions, such as guanine nucleotide exchange on small GTP-binding proteins, interaction with proteins and lipids, or enzyme inhibition. However, the function of many RLDs has not yet been elucidated [31]. Based on the crystal structure of the complex, we conclude that binding of Kti13 to Kti11 orients the latter and (partly) shields its otherwise exposed electron-carrying iron atom. This model is supported by electron transfer assays demonstrating that Kti13 alters the electron transfer abilities of Kti11. As the endogenous reductase of Kti11 has not yet been identified, these assays were performed using bacterial general rubredoxin reductase NorW, and with cytochrome c as an electron acceptor. Therefore, the results should be interpreted with caution. However, assuming similar behaviour of the endogenous Kti11 partners, we hypothesize that complexing of Kti11 with Kti13 may prevent electron transfer between Kti11 and random redox active molecules in the cell to ensure biologically relevant electron flow. According to published data, the action of Kti13 appears to be particularly important in the case of the large Elongator complex [5,17]. Kti13 may facilitate Kti11-mediated electron delivery to Elongator, either by interacting with Elongator subunit(s) or by facilitating this binding by orienting Kti11. Alternatively, Kti13 may dissociate from Kti11 when the latter binds to the Elongator complex and transfers electrons to the [4Fe–4S] cluster of Elp3. A further explanation of Kti13 function may lie in the location of its histidine residue (H297), which is located closely to the iron FEBS Journal 282 (2015) 819–833 ª 2015 FEBS


atom of Kti11. This arrangement affects the mid-point potential of the redox active protein, altering its electron transfer abilities. Therefore, Kti11 complexed with Kti13 may be a more efficient electron donor to partners with otherwise unfavourable mid-point potentials. The redox potential of iron-containing Kti11 was estimated at 6 mV [26], which is the highest value reported for rubredoxins. This property may limit the number of partners that are able to accept electron from this donor. Although no data are available regarding the potential of the [4Fe–4S] clusters of Dph2 and Elp3, if the redox potential of the Elp3 [4Fe–4S] cluster is much higher than that of Dph2, the Kti13 function of lowering the mid-point potential of Kti11 will be particularly important for functioning of the Elongator complex. Deletion of Kti13 may therefore result in inefficient, but not abolished, transfer of electrons from Kti11 to the Elongator complex, without strongly affecting diphthamide biosynthesis. This proposed hypothetical role of Kti13 in the Kti11/Kti13 complex, supported by the in vitro electron transfer analysis, may explain the results showing significantly lower levels of U34 modification [5,17] in Dkti13 cells, which is sufficient to mediate resistance to zymocin toxin that targets such modifications. It is also interesting that we observed the exclusive presence of ironbound Kti11 in Kti11–Kti13 complexes, although both zinc and iron were found to be associated with recombinant Kti11 expressed on its own [26]. We cannot exclude the possibility that zinc-bound Kti11 may play some other role(s) in the cell, unrelated to electron transfer. However, our data indicate that Kti13 complexes with Kti11 only when the latter is loaded with an iron ion. Thus, in addition to potentially controlling the redox activity and specificity of the downstream electron acceptors of Kti11, Kti13 may ensure that Kti11 is appropriately loaded with iron and thus ensure the functionality of Kti11 as electron donor. The functional versatility of the seven-bladed propeller RLD family of proteins suggests that Kti13 may also play additional roles, such as interaction with hypothetical molecules donating electrons to Kti11, and possibly other cellular functions unrelated to Elongator. Further analyses are required to identify additional Kti13 partners and elucidate how Kti13 contributes to their biological function.

Experimental procedures Plasmids and cloning Standard cloning procedures using unique restriction sites were used to introduce PCR-amplified coding sequences in



the pET24 backbone (Novagen, Madison, WI, USA). The following plasmids were constructed and used in this study: pBS5000 encoding His6-tagged Kti11, pBS5001 encoding His6-tagged Kti13, pBS5009 encoding His6-tagged TEVNorW (TEV - Recognition sequence for Tobacco Etch Virus protease), pBS5012 encoding His6-tagged Kti11/ Kti13 and pBS5103 encoding Kti11(1–57)-His6/Kti13. Additionally, the following plasmids were used for production of Kti11(1–57)-His6/Kti13 complexes comprising mutant variants of Kti13 constructed by mutagenesis on the pBS5103 or on plasmids already carrying single mutation as templates: pBS5209 (W294E), pBS5218 (H297A), pBS5220 (W294E/H297A), pBS5221 (W96A), pBS5222 (K157D), pBS5223 (W229C), pBS5224 (W96A/K157D) and pBS5225 (W96A/W229C). The sequences of oligonucleotides used in this study are available on request.

Protein production and purification Recombinant Kti11, Kti13 and NorW fused to an N-terminal hexahistidine tag (followed by a TEV recognition site in the case of NorW) were produced in E. coli under the control of a T7 promoter. The Kti11/Kti13 complex and its mutated variants were produced from bi-cistronic vectors in which genes encoding the first 57 amino acids of C-terminally His6-tagged Kti11 were followed by wildtype or mutated Kti13. The two proteins were placed under the control of a T7 promoter. Mutations within kti13 were introduced by amplification of the whole vector using PfuUltra II Phusion HS DNA polymerase (Agilent Technologies, San Diego, CA, USA) and primers carrying the desired mutation(s), followed by DpnI digestion. Proteins were expressed overnight in E. coli BL21-codonPlus (DE3)-RIL incubated at 25°C in auto-inducing medium supplemented with 50 lgmL 1 kanamycin. Frozen cell pellets were resuspended in 20 mL PBS per 1 g cells, and cell lysis was performed using a cell disruptor at 1.5 kbar. After centrifugation at 10 000 g for 30 min and filtration through a 0.45 lm filter, NaCl and imidazole concentrations were adjusted to 500 and 20 mM, respectively (except for the Kti11/Kti13 complex and its mutants). The solution was loaded onto a 1 mL HisTrapFF column (GE Healthcare Europe GmbH, Freiburg, Germany) previously equilibrated with PBS containing 500 mM NaCl and 20 mM imidazole. In the case of the Kti11/Kti13 complex and its mutants, only the imidazole concentration was adjusted to 20 mM and the column equilibrated accordingly. After washing the column with 20 mL of the same buffer, the proteins were eluted by an imidazole step gradient. Fractions containing the desired protein were concentrated using a centrifugal filter with a 3 kDa (Kti11) or 10 kDa (other proteins) molecular weight cut-off (Sartorius, Gettingen, Germany), and applied onto a HiLoad XK 16/60 Superdex 200 gel filtration column (GE Healthcare) previously equilibrated with 20 mM HE-

827


PES pH 7.5, 150 mM NaCl, 1 mM dithiothreitol. Purified proteins were concentrated as above, aliquoted, snap-frozen in liquid nitrogen, and stored at 80°C.

Crystallization, X-ray analysis and structure determination of Kti11/Kti13 complex For crystallization trials, the Kti11(1–57)-His6/Kti13 complex was concentrated to 3.5 mgmL 1, and supplemented with Tris(2-carboxyethyl)phosphine) to a final concentration of 2.5 mM. Crystallization conditions were assessed using commercially available kits from Hampton Research (Aliso Viejo, CA, US), Qiagen (Courtaboeuf, France) and Emerald Biosystems (Bainbridge Island WA, USA) using the sittingdrop vapour diffusion method in 96-well MRC-2 or MRC-3 plates (SWISSCI, Neuheim, Switzerland) at 277 and 293 K. The protein solution and precipitant solution (200 nL each) were equilibrated against 40 lL of reservoir, with all experiments being performed using a Mosquito Crystal robot (TTP Labtech, Melbourn, Hertfordshire, UK)). After optimization of the initial hit, diffraction-quality crystals were grown in 20–25% poly(ethylene glycol) 3350, 0.2 M MgCl2 with either 0.1 M HEPES pH 7.5, 0.1 M MOPS pH 7.0, or 0.1 M Tris pH 8.0. The small rod-shaped crystals started to appear from the amorphous precipitate within 3 days, and grew to their maximum size of 200 9 20 9 20 lm within 4 weeks. For native and Fe-SAD (Single-wavelength anomalous dispersion) data collection, the crystals were transferred to 35% poly(ethylene glycol) 3350, 0.2 M MgCl2, 0.1 M of the appropriate buffer for a few seconds before being flashcooled in liquid nitrogen. For Hg-SAD data collection, the crystals were transferred to reservoir solution supplemented with 10 mM thiomersal for 2 h before being transferred to the appropriate 35% poly(ethylene glycol) 3350 solution and flash-cooled in liquid nitrogen. We performed an X-ray fluorescence spectroscopy on a crystal prior to data collection. This scan showed peaks for the iron Ka and Kb edges, but only a tiny signal at the zinc Ka edge. Such traces of zinc have been shown to be produced in other cases by contamination from mother liquors. We then performed an X-ray fluorescence scan around the K absorption edges of iron and zinc. The scan showed only at the K edge of zinc (approximately 1.28 A) noise, and therefore did not indicate the presence of zinc, while the scan at the iron edge (approximately 1.74 A) showed a strong signal for iron (data not shown). A SAD at the iron K dataset was collected to a resolution of 2.4 A edge at 100 K on the Proxima 1 beamline of the synchrotron SOLEIL (Gif-sur-Yvette, France) using a Pilatus 6M detector (Dectris, Baden, Switzerland). SAD data were also collected at the mercury LIII edge from a thiomersal-deriva on the Proxima 1 beamline. Native tized crystal to 2.1 A at 100 K on the ID23–2 midata were collected to 1.45 A crofocus beamline of the European Synchrotron Radiation

828


Facility (Grenoble, France) using a Pilatus 2M detector (Dectris). All data were indexed and processed using XDS [37], and scaled using AIMLESS [38,39] from the CCP4 suite [40]. The crystals belonged to space group C2221 with unit b = 89.0 A, c = 163.5 A. Matcell dimensions a = 49.8 A, thews analysis [41] suggested the presence of one copy of 3/Da, the complex per asymmetric unit (VM = 2.03 A 39.5% solvent). The anomalous signal of the Fe peak dataset was too weak to solve the structure, but the signal from the Hg peak dataset was much stronger. The Hgbound structure was solved by SAD using AutoSol [42–45] from the PHENIX suite [46], which identified 11 mercury sites. Following phasing and density modification, AutoSol built 243 residues (61.7% of the expected residues) comprising 36 residues (57.1%) for Kti11 and 207 residues (62.3%) for Kti13. In total, 102 residues were docked into the sequence (25.9%). ARP/wARP [47] was used to improve the model, which was then used to phase the native data in MOLREP [48]. A further round of model building was performed in ARP/wARP, which built 385 residues (97.7%). The structure was refined using PHENIX.REFINE [49] and BUSTER [50], followed by iterative model building in COOT [51]. To further confirm the nature of the metal ion, two other datasets using wavelengths at either side of the iron K edge were later col on the Proxima 1 beamline of the lected to 2.1 A synchrotron SOLEIL (Gif-sur-Yvette, France) using a Pilatus 6M detector (Dectris). Using phases from the final model, two types of maps were created (data not shown). The first map is an anomalous Fourier difference map using data collected just above the absorption edge, with the highest peak corresponding to the Fe atom position (27r level). Sulfur atoms are seen at the 6r level. The second map, an ‘element-specific map’ difference Fourier map [52] was created by taking the difference of anomalous difference data from just above and below the absorption edge of iron. This ‘Fe-specific map’ map shows only one significant peak at the 10r level corresponding to the Fe position, with noise peaks emerging at the 3r level. The quality of the refined models was assessed using MOLPROBITY [53] and PROCHECK [54]. Data collection and refinement statistics are summarized in Table 1. Molecular graphics figures were generated using the PyMOL Molecular Graphics System (Schr€ odinger, New York) or the UCSF CHIMERA package (supported by US National Institute of General Medical Sciences grant number P41-GM103311) [55]. Coordinates and structure factors have been deposited at the Protein Data Bank under accession number 4X33.

Isothermal titration calorimetry ITC experiments were performed using a MicroCal iTC200 (Malvern Instruments Ltd, Malvern, Worcestershire, UK) at 10°C. The N-terminally His6-tagged Kti11 and Kti13




Table 1. Data collection, phasing and refinement statistics. Values in parentheses are for the highest-resolution shell.

Data collection statistics Diffraction source Wavelength ( A) Temperature (K) Detector Crystal to detector distance(mm) Rotation range per image (°) Total rotation range (°) Exposure time per image (s) Space group a, b, c ( A) a, b, c (°) Mosaicity (°) Resolution range ( A) Total number of reflections Number of unique reflections Completeness (%) Multiplicity {I/r(I)} Rmerge (%)a Rr.i.m. (%)b Rp.i.m. (%) CC1/2c Overall B factor from Wilson plot ( A2) Refinement statistics Resolution range ( A) Reflections used in refinement Reflections used for R-free Rwork (%)d Rfree (%)e Number of non-hydrogen atoms Protein Ion Ligand Water RMSD valuesf Bonds ( A) Angles (°) Average B factor Protein Ion Ligand Water Ramachandran plotg (%) Core Allowed

Native

Fe-SAD

Hg-SAD

ESRF ID23-2 0.873 100 Pilatus 2M 139.3

SOLEIL Proxima 1 1.7389 100 Pilatus 6M 197.2

SOLEIL Proxima 1 1.008 100 Pilatus 6M 332.5

0.05

0.2

0.2

180 0.03

200 0.2

200 0.2

C2221 49.79, 89.01, 163.53 90.0, 90.0, 90.0 0.098 44.5–1.45 (1.47–1.45) 431 552 (20 863)

C2221 49.79, 88.64, 163.21 90.0, 90.0, 90.0 0.142 44.32–2.09 (2.15–2.09) 145 383 (8261)

C2221 49.72, 89.47, 162.85 90.0, 90.0, 90.0 0.079 44.74–1.95 (1.99–1.95) 196 711 (12 686)

64 706 (3090)

21 058 (1540)

27 065 (1804)

99.8 (97.3) 6.7 (6.8) 9.5 (0.8) 9.6 (194.0) 11.4 (214.5) 4.4 (87.5) 0.998 (0.275) 16.93

96.7 (86.6) 6.9 (5.4) 13.7 (3.9) 9.0 (38.4) 10.5 (46.8) 5.4 (26.3) 0.997 (0.866) 20.89

99.7 (95.7) 7.3 (7.0) 10.1 (1.3) 10.1 (136.7) 11.8 (148.2) 5.9 (81.6) 0.997 (0.481) 33.18

21.2–1.45 (1.49–1.45) 64 580 (4649) 3209 (204) 14.97 (23.10) 17.62 (25.72) 3852 3265 10 6 571 0.01 1.09 27.2 25.13 38.62 37.73 38.69 90.3 9.1


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Table 1. (Continued). Native Generously allowed Disallowed

Fe-SAD

Hg-SAD

0.3 0.3

Rmerge = Σhkl Σi |Ii(hkl) – {I(hkl)}| / Σhkl Σi Ii(hkl). Rr.i.m. is the redundancy-independent merging R factor; Rp.i.m. is the precision-indicating merging R factor [57,58]. c CC1/2 has been defined previously [59]. Mean I/r(I) falls below 2.0 in the outer shell at 1.63 A and CC1/2 falls below 0.5 in the outer shell at 1.55 A. d Rwork = Σhkl ||Fobs| - |Fcalc|| / Σhkl |Fobs|. e Rfree = Σhkl ||Fobs| - |Fcalc|| / Σhkl |Fobs|, calculated using a random set comprising 5% of the reflections that were not included throughout refinement. f Using ideal values from Engh & Huber [60]. g The percentage of residues in Ramachandran plot areas was determined using PROCHECK [54]. a

b

proteins were purified as described above using PBS at pH 7.4 as buffer for size-exclusion chromatography. During a titration experiment, Kti13 (24 lM) stirred at 1000 rpm was injected with 20 successive 2 lL aliquots of 330 lM Kti11 at 120 s intervals. After correction for heat of dilution, integrated heat effects were analysed using the MICROCAL ORIGIN software (OriginLabs, Northampton, MA, USA). Data fitting was based on a one-site binding model, which yielded the association constant (Ka), number of binding sites on the protein (N) and the enthalpy of binding (DH). The parameters presented are mean values obtained from six independent ITC runs with two batches of Kti11 protein.

Dynamic light scattering The hydrodynamic radii of the Kti11/Kti13 complex were measured by dynamic light scattering using a DynaPro NanoStar instrument (Wyatt Technology, Santa Barbara, CA, USA). A 50 lL aliquot of protein complex at 3.5 mgmL 1 was analysed at 20°C in a JC-040 DynaPro NanoStar disposable cuvette (Wyatt Technology) after centrifugation at 14 000 g for 15 min. Ten measurements were made, with an acquisition time of 7 s each. The data were analysed using the graphical size analysis software, Dynamics, provided with the instrument.

Electron transfer analysis The electron transfer analyses were performed in buffer comprising 50 mM HEPES pH 7.5, 150 mM NaCl and 5% glycerol at room temperature in quartz cuvettes of total volume of 1 mL using a Cary 50 Bio UV/visible spectrophotometer (Varian Inc., Palo Alto, CA, USA). The reduction of Kti11 (25 lM) by NorW (1.5 lM) in the presence of NADH (285 lM) was performed aerobically. The reaction was started by addition of NADH. As Kti11 exhibits a classical rubredoxin-like spectrum [26], with one of the peaks at approximately 490 nm in its oxidized but not reduced state, the reduction of Kti11 was followed at

830

488 nm as described elsewhere [12]. Both NAD+ and NADH absorb negligibly at this wavelength. With time, re-oxidation of Kti11 by air was observed. In the case of reduction of the Kti11/Kti13 complex, dithionite was subsequently added to 1 mM final concentration. The reduction of horse heart cytochrome c (16 lM) by Kti11 (2 lM) in the presence of NorW (0.5 lM), NADH (115 lM) and Kti13 as indicated in the text (0.65, 2 or 4 lM) was followed at 550 nm. The components of the reaction were assembled in a cuvette anaerobically under argon that was subsequently sealed carefully with plastic wrap before transferring it to the spectrophotometer. The reaction was started by addition of NADH by piercing the plastic wrap with a long thin pipette tip and immediately resealing the cuvette. For reduction of cytochrome c upon prior Kti11 reduction, Kti11 (6.6 lM), NADH (115 lM) and NorW (0.5 lM) were anaerobically mixed. When Kti13 (6.6 lM) was used, it was added to the cuvette after approximately 2 min and incubated for at least 10 min to ensure formation of the Kti11/Kti13 complex. In each case, after sealing of the cuvette, the reaction was started by addition of cytochrome c (16 lM) by piercing the plastic wrap as described above, and the reaction was followed at 550 nm.

Acknowledgements We would like to thank Pierre Poussin-Courmontagne and Catherine Birck from the Structural Biology and Genomics Platform at Institut de Genetique et de Biologie Moleculaire et Cellulaire for technical support, members of our group for discussion and advice, and Institut de Genetique et de Biologie Moleculaire et Cellulaire services for their support. We also thank the members of the European Synchrotron Radiation Facility/European Molecular Biology Laboratory joint structural biology group and staff of the synchrotron SOLEIL, especially Beatriz Guimaraes and Pierre Legrand, for use of beamline facilities and for help during data collection. This work was supported by



the Ligue Contre le Cancer (Equipe Labellisee 2014), the Centre National pour la Recherche Scientifique, the Centre Europeen de Recherche en Biologie et en Medecine-Institut Genetique Biologie Moleculaire Cellulaire (CERBM-IGBMC), Project Elongator of the Agence Nationale pour la Recherche (grant ANR-13BSV8-0005-01), grant number ANR-10-LABX-0030INRT managed under the program Investissements d’Avenir ANR-10-IDEX-0002-02, as well as French Infrastructure for Integrated Structural Biology grant number ANR-10-INSB-05-01, and Instruct as part of the European Strategy Forum on Research Infrastructures.


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