Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A

Abstract

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645-33651). Here, we report two new LplA structures, LplA.lipoyl-5'-AMP and LplA.octyl-5'-AMP.apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5'-AMP reaction intermediate and rotation of the C-terminal domain by about 180 degrees . These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys(133) residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.

FIGURE 1.

Structure of the LplA·lipoyl-AMP complex. A, overall structure of the LplA·lipoyl-AMP complex. LplA is shown in ribbon form. N- and C-terminal domains and the connecting loop of LplA are in gray, cyan, and green, respectively. The lipoate-binding loop (residues 69–76) and the adenylate-binding loop (residues 165–184) are highlighted in magenta and yellow, respectively. The hydrogen bond between Ser⁷² and His¹⁴⁹ is shown by a dashed line in green. The secondary structural elements, N and C termini, and some residues shown in stick form are labeled. Lipoyl-AMP is shown in stick form in orange (bond) and atom colors. B, superposition of structures of the LplA·lipoyl-AMP complex (in cyan) and unliganded LplA (in green; Protein Data Bank code 1X2G, molC). Lipoate-binding and adenylate-binding loops in the complex are highlighted in magenta and orange, respectively, and those in unliganded LplA are in black and gray, respectively. A black ellipse and dashed line represent the 2-fold rotational symmetry axis of the C-terminal domain. C, stereo view of the LplA active site. The F_o − F_c omit electron density map for lipoyl-AMP contoured at 3σ is shown in green. Lipoyl-AMP and some residues of LplA are shown in stick form. The colors are the same as in A. An ordered water molecule is in red. Mg²⁺ introduced from solvent is in yellow. Hydrogen bonds are represented by dashed lines in blue.

FIGURE 2.

Structure of the LplA·octyl-AMP·apoH-protein ternary complex. A, ribbon form of the 2:2 heterotetramer protein structure in the asymmetric unit. LplAs (molA and molC) are shown in cyan and gray, respectively. ApoH-proteins (molB and molD) are shown in purple and violet-purple, respectively. Octyl-AMP is shown in stick form in orange (bond) and atom colors. B, the active site of the ternary complex (molA·molB). The ternary complex (colored as in A) is superimposed onto the LplA·lipoyl-AMP binary complex (in gray). Octyl-AMP (bond, orange), lipoyl-AMP (gray), Asn¹²¹ of LplA in the ternary complex (bond, cyan) and the binary complex (bond, gray), and Lys⁶⁴ (bond, purple) of apoH-protein are shown in stick form. The distance between C^α of Lys⁶⁴ of apoH-protein and the C¹ atom of the octyl moiety of octyl-AMP is shown by a dashed line in black (12.7 Å). C, interaction between LplA (cyan) and apoH-protein (purple). Lys⁶⁴ of apoH-protein (bond, orange) and hydrogen-bonding residues are shown in stick forms. Hydrogen bonds are represented by dashed lines in green. D, superimposition of the ternary complex (molA·molB, colored as in A) onto the unliganded LplA (green). The surface structure of the apoH-protein is shown in 40% transparence. Some helices of unliganded LplA are labeled.

FIGURE 3.

Electrostatic potential on the molecular surface. A, open view of the electrostatic potential on the interface between LplA (right) and apoH-protein (left). The C^α trace of apoH-protein in complex with LplA is shown in green (right). The visible part of octyl-AMP is shown in stick form in yellow (bond) and atom colors. B and C, electrostatic potential on the surface of the lipoyl domain of the E. coli pyruvate dehydrogenase complex (Protein Data Bank code 1QJO) and the α-ketoglutarate dehydrogenase complex (Protein Data Bank code 1PMR) are shown, respectively. Lys⁴¹ (K41) and Lys⁴³ (K43) are lysine residues to be lipoylated. Negatively and positively charged surfaces are shown in red and blue, respectively. Some charged residues are labeled.

FIGURE 4.

Structure-based proposal for a reaction mechanism catalyzed by LplA. The mechanism of the two successive reactions is presented with schematic illustrations of the conformational change of E. coli LplA. N- and C-terminal domains and the connecting loop of LplA are shown in yellow and gradient cyan and in green, respectively. Apoprotein is shown in magenta. Adenylate and lipoate binding loops are in orange and magenta, respectively. Ad, adenosine moiety of ATP; Lip, aliphatic tail and a dithiolane ring of lipoic acid. The dotted line, arrow, and arrow with dashed line represent the hydrogen bond, electron transfer, and proton transfer, respectively. Details of the mechanism are described under “Discussion.”