Structural basis for the binding of succinate to succinyl-CoA synthetase

Abstract

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.

Tartryl-CoA inhibits succinyl-CoA synthetase

“…4). These interactions are very similar to the interactions of two of the phosphate O atoms with GTPSCS in the complex with phosphate, Mg 2+ -succinate and CoA (PDB entry 5cae; Huang & Fraser, 2016). The two hydroxyl groups of tartryl-CoA form interactions similar to those of the third O atom of phosphate, hydrogen-bonding to N " of His259 and to the amide N atom of Gly163.…”

“…The structure solution was obtained by molecular replacement (McCoy et al, 2007) using the structure of succinatebound pig GTPSCS (PDB entry 5cae; Huang & Fraser, 2016) as the model. Ideal values for the geometry of the ligand were obtained using Grade ( Structure solution and refinement.…”

“…C atoms are shown in cyan for tartryl-CoA. (b) Phosphate, succinate and interacting residues of the protein in the structure of Mg 2+ -succinate-bound GTPSCS (PDB entry 5cae; Huang & Fraser, 2016) are drawn as stick models. C atoms are shown in green for succinate (abbreviated SIN).…”

“…The phosphate-binding site is close to the active-site histidine residue at the interface between the -subunit and -subunit (Fraser et al, 2000). The succinate-binding site lies in the carboxy-terminal domain of the -subunit, with a magnesium ion playing an essential role in binding succinate since it interacts with one carboxylate O atom of succinate, the phosphate ion and four water molecules (Huang & Fraser, 2016). The active-site histidine residue is phosphorylated during catalysis, and the phosphohistidine loop has been proposed to shuttle between the succinyl-CoAand phosphate-binding site and the nucleotide-binding site to transfer the phosphoryl group (Fraser et al, 1999).…”

Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

“…S3E-H, table S3 and S4). Amongst these were several proteins known to interact with both metabolites, such as Uridine-cytidine kinase (UCK1 and UCK2), casein kinase (CSNK1A1, CSNK1D, CSNK2A1) and succinyl CoA synthetase (SUCLA2 and SUCLG2) [23][24][25][26][27] . Importantly, we identified proteins not previously reported to have this property, such as, ATP binding cassette super family proteins (ABCE1, ABCF1, etc.…”