ADP-Binding Site of Escherichia coli Succinyl-CoA Synthetase Revealed by X-ray Crystallography

• Published in: Biochemistry (Easton) Vol. 39; no. 1; pp. 17 - 25
• Main Authors: Joyce, Michael A, Fraser, Marie E, James, Michael N. G, Bridger, William A, Wolodko, William T
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.01.2000
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Binding Sites; Computer Simulation; Crystallization; Crystallography, X-Ray; Escherichia coli; Escherichia coli - enzymology; Histidine - analogs & derivatives; Histidine - metabolism; Magnesium - chemistry; Magnesium - metabolism; Models, Molecular; Phosphorylation; Protein Structure, Secondary; Protein Structure, Tertiary; Succinate-CoA Ligases - chemistry; Succinate-CoA Ligases - metabolism; succinate-CoA-ligase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi991696f

Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction via a phosphorylated histidine intermediate (His 246α):  succinyl-CoA + Pi + NDP ↔ succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of phosphorylated Escherichia coli SCS were soaked in successive experiments adopting progressive strategies. In the first experiment, 1 mM ADP (>15 × K d) was added; Mg2+ ions were omitted to preclude the formation of an insoluble precipitate with the phosphate and ammonium ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R working = 17.2%, R free = 22.8% for data to 2.9 Å resolution). Catalysis requires Mg2+ ions; hence, the “true” nucleotide substrate is probably an ADP−Mg2+ complex. In the successful experiment, the phosphate buffer was exchanged with MOPS, the concentration of sulfate ions was lowered, and the concentrations of ADP and Mg2+ ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP−Mg2+ complex bound in the ATP-grasp fold of the N-terminal domain of each β-subunit (R working = 19.1%, R free = 24.7% for data to 3.3 Å resolution). We describe the specific interactions of the nucleotide−Mg2+ complex with SCS, compare these results with those for other proteins containing the ATP-grasp fold, and present a hypothetical model of the histidine-containing loop in the “down” position where it can interact with the nucleotide ∼35 Å from where His 246α is seen in both phosphorylated and dephosphorylated SCS.