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 mutation 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 chaperone 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 BIOCHEMISTRY 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 + BIOPHYSICS AND 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 COMPUTATIONAL BIOLOGY 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, r