Crystal structure of bacterial succinate:quinone SdhA in complex with its assembly factor SdhE

Megan J. Mahera,1, Anuradha S. Heratha, Saumya R. Udagedaraa, David A. Dougana, and Kaye N. Truscotta,1

aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia

Edited by Amy C. Rosenzweig, Northwestern University, Evanston, IL, and approved February 14, 2018 (received for review January 4, 2018) Succinate:quinone oxidoreductase (SQR) functions in energy me- quinol:FRD), respectively (13, 15). The importance of this tabolism, coupling the tricarboxylic acid cycle and electron transport family, in normal cellular , is manifested by the iden- chain in bacteria and mitochondria. The biogenesis of flavinylated tification of a in human SDHAF2 (Gly78Arg), which is SdhA, the catalytic subunit of SQR, is assisted by a highly conserved linked to an inherited neuroendocrine disorder, PGL2 (10). Cur- assembly factor termed SdhE in bacteria via an unknown mecha- rently, however, the role of SdhE in flavinylation remains poorly nism. By using X-ray crystallography, we have solved the structure understood. To date, three different modes of action for SdhE/ of Escherichia coli SdhE in complex with SdhA to 2.15-Å resolution. Sdh5 have been proposed, suggesting that SdhE facilitates the Our structure shows that SdhE makes a direct interaction with the binding and delivery of FAD (13), acts as a for SdhA flavin adenine dinucleotide-linked residue His45 in SdhA and main- (10), or catalyzes the attachment of FAD (10). Moreover, the re- tains the capping domain of SdhA in an “open” conformation. This quirement for SdhE in SdhA biogenesis remains controversial, as re- displaces the catalytic residues of the ac- cent studies have demonstrated that flavinylation of bacterial, archaeal, tive site by as much as 9.0 Å compared with SdhA in the assembled and mitochondrial SdhA can still occur in the absence of the assembly SQR complex. These data suggest that bacterial SdhE , and factor (16–19). In this study, we have determined the crystal structure their mitochondrial homologs, are assembly chaperones that con- of the Escherichia coli flavinylation factor SdhE in complex with its strain the conformation of SdhA to facilitate efficient flavinylation client protein SdhA to 2.15-Å resolution. This three dimensional while regulating succinate dehydrogenase activity for productive structure of an SQR assembly intermediate provides valuable insights biogenesis of SQR. into the evolutionary conserved process of flavoprotein assembly.

SdhE | flavinylation | structure | SdhA | assembly Results and Discussion Structure of SdhA in Complex with Its Assembly Factor SdhE. To obtain uccinate:quinone oxidoreductase (SQR) is a multisubunit E. coli SdhA in complex with its assembly factor E. coli SdhE, we membrane-associated found in the cytoplasm of bacteria coexpressed untagged recombinant SdhA together with recombi- S + 2 and in the matrix of mitochondria (where it is commonly termed nant His6-tagged SdhE in E. coli (Fig. 1A). By using Ni -affinity complex II). The enzyme is central to cellular metabolism and chromatography, we isolated a mixture of free His6-SdhE and energy conversion, contributing to the tricarboxylic acid cycle and the . It catalyzes the oxidation of suc- Significance cinate to fumarate, which is coupled to electron transfer through flavin adenine dinucleotide (FAD) and three Fe–S clusters, resulting Assembly factors play key roles in the biogenesis of many multi- in the reduction of the electron carrier ubiquinone to ubiquinol. The subunit protein complexes regulating their stability, activity, or overall architecture of the bacterial and mitochondrial complexes is incorporation of essential cofactors. The bacterial assembly factor highly conserved, with both forming a heterotetrameric SdhE (also known as Sdh5 or SDHAF2 in mitochondria) promotes protein complex (1, 2). The catalytic flavoprotein (SdhA in bacteria, covalent attachment of flavin adenine dinucleotide (FAD) to SdhA Sdh1 in yeast, and SDHA in humans) contains a for and hence the assembly of functional succinate:quinone oxidore- dicarboxylic acid and an N3-histidyl-8α-FAD linkage (1, 2). The ductase (also known as complex II). Here, we present the crystal covalent attachment of FAD to SdhA is essential for the oxidation of structure of Escherichia coli SdhE bound to its client protein SdhA. succinate by the enzyme (3). In the mature complex, SdhA connects This structure provides unique insight into SdhA assembly, to the inner membrane through interaction with the Fe–Sprotein whereby SdhE constrains unassembled SdhA in an “open” con- SdhB, which makes direct contact to both integral membrane sub- formation, promoting covalent attachment of FAD, but renders units, SdhC and SdhD. Ubiquinone binds at the interface of SdhB the holoprotein incapable of substrate . These data also and the two membrane proteins (SdhC and SdhD), with the cofac- provide a structural explanation for the loss-of-function mutation, tors in SQR forming a direct path (∼40 Å long) for the transfer of Gly78Arg, in SDHAF2, which causes hereditary 2. electrons into the respiratory chain (2). Significantly, loss of complex II activity in humans is associated with and tumor Author contributions: M.J.M., D.A.D., and K.N.T. designed research; M.J.M., A.S.H., and S.R.U. performed research; M.J.M., A.S.H., S.R.U., D.A.D., and K.N.T. analyzed data; and syndromes including hereditary paraganglioma (PGL) (4–8). M.J.M., D.A.D., and K.N.T. wrote the paper. The assembly of SQR is assisted by several assembly factors The authors declare no conflict of interest. involved in biogenesis and regulation of assembly in- This article is a PNAS Direct Submission. termediates (9–12). The most widely conserved SQR assembly Published under the PNAS license. factor (10, 13, 14) is termed SdhE in bacteria [also known as Sdh5 Data deposition: The atomic coordinates and structure factors have been deposited in the in yeast and SDH assembly factor 2 (SDHAF2) or SDH5 in hu- Protein Data Bank, www.wwpdb.org (PDB ID code 6C12). Saccharomyces cerevisiae mans]. Initially characterized in , Sdh5 is 1To whom correspondence may be addressed. Email: [email protected] or essential for the assembly of active complex II promoting the co- [email protected]. valent attachment of FAD to Sdh1 (10). Likewise, bacterial SdhE is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. also required for SQR and fumarate reductase (FRD) activity 1073/pnas.1800195115/-/DCSupplemental. promoting flavinylation of SdhA and FrdA (subunit A of the Published online March 7, 2018.

2982–2987 | PNAS | March 20, 2018 | vol. 115 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1800195115 Downloaded by guest on September 23, 2021 (Fig. 1D). To elucidate the atomic structure of the SdhAE protein assembly, we crystallized the complex and determined its structure to 2.15-Å resolution by X-ray crystallography (Fig. 2A and Table S1). The crystal contains two almost identical SdhAE complexes in the asymmetric unit. The final SdhAE model includes residues 1–583 from the SdhA protein, apart from two regions: residues 52–67 and 107–137 in addition to residues 2–83 from the SdhE protein. The structure of SdhA is composed of four domains (20): an FAD-binding domain (residues 1–245 and 351–431), which in- cludes a Rossmann-type fold and provides the binding site for FAD (Fig. 2A, pink); a capping domain composed of residues 245–351 (Fig. 2A, gray); a helical domain composed of residues 431–547 (Fig. 2A, blue); and a C-terminal domain composed of residues 547–583 (Fig. 2A, orange). FAD was well resolved in the electron density map and, consistent with the in-gel assay (Fig. 1D), was covalently attached to SdhA via a N3-histidyl-8α-FAD linkage (1.6 Å) between the Ne2 atom of His45 and the C8 atom of the isoalloxazine group (Fig. 2B). Although a dicarboxylate is required for the flavinylation reaction (21), the SdhAE structure presented here captures the assembly intermediate after flavinylation has occurred. Consistently, there was no evidence in the electron density for the presence of a dicarboxylate associated with SdhA. SdhE forms a single compact domain (Fig. 2A, green) composed of five α-helices and interfaces closely with SdhA, where it is wedged between the FAD and capping domains. The SdhE protein forms an intimate complex with SdhA, with 2 A–C Fig. 1. Isolation of the binary protein complex containing SdhE, SdhA, and a buried surface area of 1,455 Å (Fig. 3 ). Three regions of – covalently bound FAD. (A) Schematic illustration showing His6-tagged SdhE SdhE make contact with the SdhA protein, namely residues 5 25 and untagged SdhA constructs used in this study. The numbers reflect amino (which encompass helix α1, the N terminus of α2, and the loop that acid residues of the authentic portion of the protein constructs. (B) Elution connects these two regions), residues 47–61 (which form helix α4), profile (Left) showing separation of the SdhAE complex (first peak) from free and residues 80–86 (which form the C terminus; Fig. 3A and Fig. SdhE (second peak) using size-exclusion chromatography. (Right) Confir- S1). Overall, the interface is composed of electrostatic interactions, mation of purified SdhAE complex by Coomassie Brilliant Blue (CBB)-stained 18 hydrogen bonds, and hydrophobic contacts and exhibits a high SDS/PAGE. (C) Elution profile of purified SdhAE relative to indicated protein degree of charge complementarity (Fig. 3D). Importantly, the in- standards analyzed by size-exclusion chromatography. The molecular weight of the SdhAE complex is estimated to be 88.5 kDa. (D) Detection of FAD terface defined by the SdhAE structure is consistent with a recent fluorescence (Fluor.) in protein samples (Upper)ofE. coli lysate (lane 1) and biochemical study that identified two SdhE residues (Arg8 and purified SdhAE complex (lanes 2 and 3) following separation by SDS/PAGE. Met17), which could be photocrosslinked to SdhA or FrdA (18). (Lower) The same gel following CBB staining. Only the region of the gel Consistent with an important functional role for residues located containing SdhA is shown. within this interface, mutagenesis of Arg68 and Tyr71 in yeast Sdh5 (22) (equivalent to Arg8 and Trp11 in E. coli SdhE) prevented Sdh1 flavinylation. Similarly, the inherited mutation coding for His6-SdhE in complex with untagged SdhA. A homogenous prepa- Gly78Arg in human SDHAF2 (equivalent to Gly16 in E. coli SdhE, ration of the SdhA/SdhE (SdhAE) complex was then purified by which is located in the loop between the α1andα2 helices; Fig. S1) size-exclusion chromatography (Fig. 1B). The SdhAE complex strongly destabilizes the SDHAF2 interaction with SDHA (10, 23). eluted from an analytical size-exclusion column at a volume corre- Collectively, these data suggest that the loop that connects the sponding to the molecular weight of a heterodimer (Fig. 1C)and α1andα2 helices of SdhE plays a crucial role in the stability of FAD was covalently bound (to SdhA) within the binary complex (10) the complex. SdhA interacts with the SdhE protein in three main BIOCHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 2. Structure of the SdhAE complex. (A) The SdhE protein (green) forms a 1:1 complex with SdhA. SdhE is positioned between the SdhA FAD-binding (pink) and capping (gray) domains. The helical and C-terminal domains of the SdhA protein are shown in pale blue and orange, respectively. FAD (yellow spheres) is covalently bound to the SdhA protein. (B) In the SdhAE complex, residue His45 (SdhA; pink) is covalently bonded to the FAD cofactor (yellow). The carbonyl group of SdhE residue Gly16 (green) participates in a hydrogen bonding interaction with SdhA His45.

Maher et al. PNAS | March 20, 2018 | vol. 115 | no. 12 | 2983 Downloaded by guest on September 23, 2021 capping domain, coupled with the apparent mobility of residues 52–67 and 107–137, exposes the isoalloxazine group of the bound FAD in the SdhAE complex to solvent (solvent-accessible surface area is 64 Å2;Fig.4B), whereas the cofactor is completely buried in the intact SQR structure [PDB ID code 2WDQ (24); solvent accessible area is 5 Å2;Fig.4C]. Interestingly, in contrast to the SdhAE structure, in which the FAD binding site remains exposed in the presence of covalently bound FAD, the FAD binding pocket of most FAD-bound enzymes is enclosed. For example, the incorporation of FAD into L-aspartate oxidase triggers structural reorganization of the capping domain relative to the FAD- binding domain (26). In this case, the apo protein exists in an open conformation in which both domains are well separated. However, upon binding of FAD, the capping domain rotates 27° toward the FAD-binding domain, thereby closing the binding site. This type of “closed” conformation is commonly observed in these types of enzymes following FAD incorporation (27–29). Hence, our FAD-bound struc- ture of SdhA in complex with SdhE is unique in presenting an FAD- bound, open arrangement of the FAD-binding and capping domains, and helps to reveal the role of SdhE in stabilizing this conformation.

Direct Contact Between SdhE and the Flavin Binding Site of SdhA Facilitates Covalent Flavinylation. In contrast to the significant struc- tural rearrangements in SdhA, which accompany formation of the

Fig. 3. SdhE forms an intimate contact with SdhA. (A) The SdhE protein is rainbow-colored (blue at the N terminus to red at the C terminus). Secondary structural elements are labeled (helices α1−α5). The capping domain of SdhA is colored gray and the remainder of SdhA is colored pink. The FAD cofactor is represented as yellow spheres. (B) Surface representation of the SdhAE complex with SdhE shown in cyan and SdhA in dark blue. The FAD cofactor is represented as yellow sticks. (C) “Open book” representation of the SdhAE complex from B (SdhE, cyan; SdhA, dark blue). The protein surfaces that are buried in the complex are highlighted in white. (D) The SdhAE complex as represented in C. The protein surfaces are colored according to the elec- trostatic potentials (red, negatively charged; blue, positively charged; white, uncharged). The protein–protein interface in the SdhAE complex shows charge complementarity.

regions: residues 38–50, 138–143, and 210–217 of the FAD domain (which frame the FAD binding site); residues 247–251 of the upper surface of the capping domain; and residues 505–515 at the tip of the helical region of the FAD domain (nomenclature defined in ref. 18). Consistent with the SdhAE structure, FrdA residues 456– 462, which are equivalent to the helical region in SdhA, were shown to photo–cross-link to Met17 of SdhE (18).

Rotation of the SdhA Capping Domain Accompanies Formation of the SdhAE Complex. Superposition of SdhA from the SdhAE complex with SdhA from the intact SQR enzyme [Protein Data Bank (PDB) ID code 2WDQ; chain A (24)] reveals a number of significant dif- Fig. 4. The position of the SdhA capping domain is markedly different in ferences between the two structures (rmsd of 4.14 Å for 536 com- the SdhAE assembly relative to SQR. (A) Comparison of the orientation of mon Cα positions). Although most of the structure of the FAD- the SdhA capping domain in the structure of SQR (PDB ID code 2WDQ; red) binding domain is similar in both states, there are significant (24) with that in the SdhAE complex (blue). In SdhAE, the capping domain is changes to three distinct regions of SdhA, specifically that residues observed in an orientation rotated 43.5° away from the FAD-binding do- 40–50 are displaced by as much as 7 Å, residues 88–105 are dis- main. The FAD-binding, helical, and C-terminal domain structures for the placed by as much as 8 Å, and the capping domain (residues 246– SdhA protein within SdhAE and SQR complexes are shown in gray. The FAD cofactor is represented as yellow sticks. (B) Surface representation of the 350) is displaced by as much as 13 Å. In the SdhAE complex, the SdhAE complex (SdhE, green; SdhA capping domain, gray; FAD-binding, capping domain of SdhA is rotated away from the FAD-binding helical, and C-terminal domains, pink; FAD cofactor, yellow spheres). (C) domain by 43.5° [as analyzed by DynDom (25)] relative to that Surface representation of the SdhAB structure within SQR (24) (PDB ID code observed for the intact SQR enzyme (Fig. 4A). The rotation of the 2WDQ; SdhB, green; SdhA colored as in B).

2984 | www.pnas.org/cgi/doi/10.1073/pnas.1800195115 Maher et al. Downloaded by guest on September 23, 2021 SdhAE complex, the structure of SdhE is virtually unchanged. Su- perposition of the structure of SdhE alone (PDB ID code 1X6I) (30) with SdhE in the SdhAE complex reveals an rmsd of 0.39 Å (for 75 common core Cα atoms), with minor structural rear- rangements localized to the C terminus. Interestingly, the SdhE loop, between helices α1andα2, does not change conformation upon complex formation, despite its central location at the SdhAE interface. This part of the SdhE structure appears to be tailored for its interaction with SdhA and therefore may contribute directly to catalysis of SdhA flavinylation. Nevertheless, the autocatalytic flavinylation of several FAD-dependent enzymes has been well described in the literature. The mechanism of catalysis is proposed to involve five features (31): (i) abstraction of a proton from the FAD C8 methyl group (by bases near the residues that tether Fig. 5. The influence of bound SdhE on catalytic residues in SdhA. Super- the flavin moiety); (ii) stabilization of the negative charge at the = iii position of the SdhA proteins from the SdhAE (gray) and SQR [PDB ID code N1-C2 O2 of the isoalloxazine moiety; ( )attackby 2WDQ (24); blue] structures. For clarity, the FAD cofactor from the SdhAE the histidyl-imidazolyl group at C8α to form a covalent bond be- structure only is represented in yellow. residues (labeled) are tween the polypeptide chain and the reduced flavin; (iv)pro- displaced by as much as 9.0 Å away from the substrate binding site in the tonation of the negative charge at the N5 position of the reduced SdhAE complex relative to SQR. isoalloxazine ring system by a neighboring amide group; and (v)

reoxidation of the reduced flavin by electron transfer to . 2 As detailed in Table S2, the majority of the crucial interactions that SdhB interface in the SQR structure (2,400 Å )(24)issignificantly greater than the interface between SdhA and SdhE in the SdhAE support these steps are maintained not only in the final structures of 2 bovine and E. coli enzymes but also in the SdhAE complex de- complex (1,455 Å ), it is likely that the binding affinity of flavinylated scribed here. Therefore, these data suggest that SdhE does not play SdhA is stronger for SdhB than it is for SdhE (33). Displacement a direct role in the catalysis of SdhA flavinylation, but rather sup- of SdhE therefore likely occurs via competition with SdhB after ports a conformation of SdhA that facilitates autocatalysis. the covalent attachment of the FAD, which allows “closure” of the Part of the interaction network between SdhA and SdhE in- capping and FAD-binding domains and repositioning of the catalytic cludes a between the carbonyl oxygen of Gly16 in residues, resulting in activation of the enzyme. SdhE and the Nδ1 atom of SdhA residue His45, which is covalently bound to the FAD cofactor (Fig. 2B). This glycine residue is highly Mechanism of Action of SdhE in the Biogenesis of Holo-SdhA. Recently, conserved in SdhE/Sdh5 proteins across a broad range of species it was proposed that SdhE docks onto its target flavoprotein fol- including bacteria, fungi, plants, and animals (Fig. S1B) (14, 32). lowing closure of the capping domain over noncovalently bound Significantly, a point mutation at the equivalent Gly residue in FAD, and, as such, maintains the flavoprotein in the closed state human mitochondrial SDHAF2 (Gly78Arg) is associated with to facilitate flavinylation (18, 19). In contrast to this recently PGL2 , and this mutation dramatically impairs its in- proposed model, our structure clearly demonstrates that SdhE, in teraction with human SDHA (10, 23). Consistent with an impor- separating the FAD-binding and capping domains, holds holo- tant role for this highly conserved Gly residue in the function of SdhA in an open conformation. Therefore, we propose an alter- SDHAF2, tumor cells isolated from patients with PGL2 contain native pathway, at least for the biogenesis of holo-SdhA (Fig. 6). extremely low levels of flavinylated SDHA (10). Similarly, the In the absence of SdhE, FAD binds noncovalently to the FAD equivalent mutation (Gly16Arg) in bacterial SdhE inhibits flavi- binding domain of folded apo-SdhA. At this point, in the presence nylation of SdhA (32). Therefore, we propose that the hydrogen of dicarboxylate, spontaneous flavinylation may occur (Fig. 6, path I); bond formed between SdhE residue Gly16 and SdhA residue however, under certain conditions and in certain cell types, this His45 is crucial for efficient flavinylation of SdhA. This interaction process is very inefficient (10, 13, 18), possibly because of non- ensures that the Nδ1 atom at SdhA residue His45 is protonated, productive pathways of misfolding or unregulated assembly of promoting nucleophilic attack by the deprotonated Ne2atomat apo-SdhA (Fig. 6, path III). In path II, SdhE and FAD bind to FAD C8α to facilitate covalent bond formation between the folded apo-SdhA sequentially (as illustrated in Fig. 6) or simul- polypeptide chain and the reduced flavin. This hydrogen bond taneously. The docking of SdhE to SdhA not only maintains the may also lock the side chain of His45 in an orientation conducive capping domain of SdhA in an open conformation but also fa- to this reaction. cilitates the formation of a critical hydrogen bond between the carbonyl oxygen of SdhE residue Gly16 and the Nδ1atomof BIOCHEMISTRY SdhE Stabilizes SdhA in a Nonactive Conformation During Assembly. SdhA residue His45, which stabilizes SdhA in a state that is con- The SdhAE complex also reveals that flavinylation of SdhA does ducive to highly efficient autocatalytic flavinylation. The limited not trigger release of SdhE. Instead, SdhE is wedged between the efficiency of flavinylation in the absenceofSdhE(18)ismostlikely FAD-binding and capping domains of SdhA, relocating its active the result of the loss of exclusive protonation of the Nδ1atomand site residues (24). Specifically, the side chains of SdhA residues tethering of the side chain of His45 in SdhA that forms the N3 His242, Thr254, Glu255, and Arg286 are displaced by distances -histidyl-8α-FAD linkage. Following flavinylation of SdhA, SdhE of as much as 9.0 Å away from the dicarboxylate-binding site (in remains bound to SdhA, thereby stabilizing holo-SdhA in an in-

comparison with their positions in the SQR structure) (24), active conformation by displacement of catalytic residues (required BIOPHYSICS AND for oxidation of succinate). This is likely important to prevent elec-

which is located between the isoalloxazine ring of FAD and the COMPUTATIONAL BIOLOGY capping domain (2, 24) (Fig. 5). We propose that the significant tron leakage and hence oxidative damage in the cell. Such a role may positional displacement of these residues during SdhAE assembly be particularly important under aerobic conditions in E. coli in which prevents oxidation of succinate by the complex. This disruption of SQR is expressed (ref. 34 and references therein). Finally, we the active site is likely important in preventing electron “leakage” speculate that holo-SdhB, by virtue of a higher affinity for SdhA, from free SdhA (via succinate dehydrogenase activity) and hence the displaces SdhE from SdhA, allowing the capping domain of SdhA subsequent production of damaging reactive oxygen species. So how to close over the FAD binding domain. Within this model, we does SdhA progress in the assembly pathway and associate with the cannot exclude the possibility that other factors act to passively iron–sulfur protein SdhB? Given that the surface area of the SdhA/ or actively displace SdhE from SdhA before SdhB docking.

Maher et al. PNAS | March 20, 2018 | vol. 115 | no. 12 | 2985 Downloaded by guest on September 23, 2021 Fig. 6. Proposed SdhA assembly pathway. Path I represents flavinylation of SdhA occurring in the absence of SdhE. In this case, the yield of flavinylated SdhA and hence active SQR is very low. Path II represents SdhE-assisted flavinylation of SdhA, the optimal route for efficient flavinylation and SQR activity. Path III also represents assembly of SdhA in the absence of SdhE. In this case, however, FAD is absent or remains associated with SdhA noncovalently. This leads to limited progression in the pathway or assembly of a nonfunctional SQR. This model is based largely on the structural (this study) and cellular studies of E. coli SdhA flavinylation and assembly under aerobic conditions. CAP, capping domain of SdhA; FBD, FAD binding domain of SdhA; E, SdhE; G16, Gly16 residue in SdhE; H45, His45 residue in SdhA. FAD is indicated in yellow.

Intriguingly, mitochondria contain an additional assembly factor, interface within the FrdA–SdhE complex is smaller than that in Sdh8 (also known as SDHAF4), which forms a stable complex with the SdhAE structure (1,085 and 1,455 Å2, respectively). Signifi- flavinylated SdhA and seemingly facilitates its assembly with SdhB cantly, the structure of the two FrdA–SdhE assemblies within the (11). Nevertheless, a bacterial homolog (or functional equivalent) of asymmetric unit are nonidentical, and the average B-factor for the Sdh8 has yet to be described. The assembly intermediate SdhAB SdhE protein is approximately twice that of the FrdA protein, subsequently associates with membrane-integrated SQR subunits to indicating significant disorder. The authors also reported that the form the functional complex. In summary, SdhE has a dual function electron density for SdhE was “weaker” than for the FrdA subunit to promote efficient flavinylation of SdhA while simultaneously (35). In contrast, the crystal structure of both SdhAE complexes neutralizing its succinate dehydrogenase activity during assembly. per asymmetric unit are almost identical, with consistent average B-factors across the SdhA and SdhE proteins and ordered elec- Comparison with the E. coli FrdA–SdhE Complex. While this manu- tron density for the entire SdhE molecules (Table S1). Whether script was under review, the crystal structure of E. coli SdhE cross- these differences are a result of the different experimental methods – linked to flavinylated FrdA (denoted here as the FrdA SdhE used to generate these complexes for structural analyses will await complex) was reported to 2.6-Å resolution by Iverson and co- further investigation. workers (35). The FrdA–SdhE structure shares a number of fea- tures with that of SdhAE reported here: (i) SdhE is located Materials and Methods between the FAD and capping domains of the flavinylated protein Protein Expression, Purification, and Characterization. The SdhE and SdhA coding in both structures, and makes a direct hydrogen bonding contact sequences were amplified by PCR by using E. coli (MC4100) genomic DNA and between Gly16 (in SdhE) and the histidyl FAD linkage in the primer pairs (Bam_E, 5′-TGTAGCGGATCCGGACATTAACAACAAAGCCCG-3′; respective flavoprotein; (ii) the SdhE binding site on the flaviny- E_Hind3, 5′-CTATCGAAGCTTAAGCTTAGATTGCCACAGGACCA-3′; and Nde_A, lated protein overlaps with that of the FrdB/SdhB subunit within 5′-ATGCGACATATGAAATTGCCAGTCAGAG-3′; Xho_A, 5′-TAGCACCTCGAGT- ′ the final complex; and (iii) complex formation with SdhE ac- TAGTAAGTACGAATCTTCGG-3 ; restriction enzyme recognition sites are in- companies a rotation of the appropriate capping domain. Im- dicated in bold) and cloned into pETDuet-1. His6-SdhE and SdhA were coexpressed for 4 h at 37 °C in E. coli strain BL21(DE3) CodonPlus-RIL (Stra- portantly, comparison of the two structures also reveals a number tagene) transformed with sequence-verified pETDuet-1/His-sdhE/sdhA. Over- of significant differences, which may reflect the relative stabilities expressed His6-SdhE/SdhA was isolated from soluble lysate [in 20 mM Tris·HCl, of the protein complexes and/or the methods used to generate pH 8.0, 200 mM KCl, 10% (vol/vol) glycerol, 1 mM EDTA, 0.05% (vol/vol) Triton X-

them. The SdhAE structure reported here represents a stable 100, 0.1 mg/mL lysozyme, 5 mM MgCl2,10μg/mL DNase I] by affinity chro- complex, which was recovered following size-exclusion chroma- matography by using Ni-NTA agarose (Qiagen) under standard conditions. tography. In contrast, the FrdA–SdhE structure required the Excess SdhE was separated from the SdhAE binary complex by using a generation of a FrdA–SdhE complex by chemical cross-linking, Superdex 200 HiLoad 16/60 column (GE Healthcare) run at 0.5 mL/min in GF and, hence, as suggested by Iverson and coworkers, “reflects an buffer (50 mM Tris·HCl, pH 8.0, 300 mM NaCl). The molecular weight of the imperfectly stabilized complex” (35). Interestingly, our compari- purified SdhAE complex was estimated by size-exclusion chromatography by son of the two structures reveals that the positioning of SdhE using a Superdex 200 Increase 10/300 GL column (GE Healthcare) calibrated by – using a gel filtration protein standard mix (Bio-Rad). To analyze flavinylation, differs. In the FrdA SdhE complex, SdhE is tilted toward the an in-gel fluorescence detection method was used (10). proposed position of the cross-link. The result is a smaller rotation – of the FrdA capping domain (10.6 10.8°, dependent on whether Protein Crystallization and Data Collection. SdhAE was crystallized at 20 °C by malonate or acetate is present in the dicarboxylate binding site) in hanging-drop vapor diffusion with drops consisting of equal volumes (1 μL) the FrdA–SdhE complex compared with the SdhAE complex of protein (6.3 mg/mL in 50 mM Tris·HCl, pH 8.0, 300 mM NaCl) and crys-

(rotation of 43.5°). In addition, the size of the protein–protein tallization solution [0.1 M Hepes, pH 7.5, 0.2 M MgCl2.6H2O, 30% (wt/vol)

2986 | www.pnas.org/cgi/doi/10.1073/pnas.1800195115 Maher et al. Downloaded by guest on September 23, 2021 PEG 3350, and 40 mM NaF]. Crystals were cryoprotected in reservoir solution diffraction data (39) interspersed with manual inspection and correction with 30% (vol/vol) glycerol before flash-cooling in liquid nitrogen. Diffrac- against calculated electron density maps by using COOT (40). The refinement

tion data were collected on an ADSC Quantum 315r detector at the Aus- of the model converged with residuals R = 0.199 and Rfree = 0.258 (Table S1). tralian Synchrotron on beamline MX2 at 100 K and were processed with The structure was judged to have excellent geometry as determined by HKL2000 (36). Unit cell parameters and data collection statistics are pre- MOLPROBITY (41) (Table S1). sented in Table S1. ACKNOWLEDGMENTS. Aspects of this research were undertaken on the Structure Solution and Refinement. The crystal structure of SdhAE was solved Macromolecular Crystallography beamline MX2 at the Australian Synchrotron by molecular replacement by using PHASER (37) with search models from the (Victoria, Australia), and we thank the beamline staff for their enthusiastic and crystal structures of the SdhA subunit from the E. coli SQR [chain A; PDB ID professional support. This work was funded by Australian Research Council code 2WDQ (24)] and the E. coli “hypothetical protein” YgfY [now known to Grants DP150104639 (to K.N.T. and D.A.D.), a La Trobe University Full Fee be SdhE; PDB ID code 1X6I (30)]. The resulting model was refined by iterative Research Scholarship (to A.S.H.), and a La Trobe University Postgraduate cycles of refinement with REFMAC (38) against anisotropically corrected Research Scholarship (to A.S.H.).

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