Structure of a putative lipoate protein ligase from Thermoplasma acidophilum and the mechanism of target selection for post-translational modification

Abstract

Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A resolution. The protein was found to fall into the alpha/beta class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T.acidophilum ligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T.acidophilum structure; tryptic cleavage of the corresponding loop in the E.coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a C-terminal domain found in its counterparts in E.coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E.coli and S.pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T.acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.

Figure 1. Amino acid sequence alignment of the E. coli (E.c), T. acidophilum (T.a) and human lipoate protein ligases.

The secondary structure elements of the E. coli LplA enzyme are highlighted.

Figure 2. Structure of the LplA of T. acidophilium with lipoic acid bound at the active site.

The red colouring denotes the boundaries of the disordered region that comprises residues 179–193. The lipoic acid is depicted in sticks and designated with an arrowhead. The helices of interest are numbered H1 and H2; the β-strands of interest are numbered B4, B7 and B8.

Figure 3. Lipoic acid binding in the LplA of T. acidophilum.

(a) 2F_o—F_c map of electron density from a crystal of the native LplA soaked with R,S-lipoic acid. Lipoic acid can be seen forming interactions with Arg72 which itself forms a hydrogen bond with the carbonyl oxygen of Gly77. This interaction is shown by the broken red lines. The bond distances to the carbonyl oxygen are 3.06 and 2.86 Å. (b) Amino acid residues in the lipoic acid binding site that are highly conserved in LplAs. The residue numbers are shown in black using T. acidophilum LplA numbering. (c) Comparison of the proposed lipoic acid-binding sites in the T. acidophilum and E. coli lipoate protein ligases. The T. acidophilum protein and lipoic acid are coloured green whereas the E. coli protein and lipoic acid are coloured blue.

Figure 4. (a) The catalytic domain of E. coli BirA with biotin bound (all in blue) superimposed on the LplA of T. acidophilum with lipoic acid bound (all in green).

The boundaries of a disordered loop region in the BirA are indicated in purple colouring and marked by black asterisks. The boundaries of an equivalent disordered loop in the T. acidophilum LplA are indicated in red; the C-terminal end of the LplA loop lies close in space to the C-terminal end of the BirA loop (asterisked), whereas the N-terminal end is located at the end of an a-helix on the other side of the lipoic acid-binding site. A region in BirA that becomes ordered upon biotin binding is coloured yellow and the loop in the T. acidophilum LplA that contains Gly77 is coloured orange. (b) A higher magnification view of (a) showing the close alignment of the lipoic acid and biotin in their respective binding sites plus the almost identical positions of the nearby loop regions and conserved lysine residue near the switch point region in BirA and LplA. The colour coding is the same as in (a).

Figure 5. Limited proteolysis of E. coli LplA with trypsin.

The protein was treated with varying concentrations of trypsin at pH 7.5 and 0 °C and the products were subjected to SDS-PAGE. The left-hand lane shows LplA with no trypsin added, the other seven lanes represent the effects of trypsin at the individual concentrations shown.

Figure 6. The trypsin cleavage sites in E. coli LplA mapped onto the amino acid sequence.

The loop that was excised by trypsin is shown in red. The dotted box indicates a sequence at the N-terminal end of the lower molecular mass fragment that was protected from excision by the binding of lipoic acid.

Figure 7. Superimposition of the structures of T. acidophilum LplA (green) and E. coli LplA (blue), highlighting the extra domain in the E. coli enzyme.

Figure 8. Domain structure of lipoate protein ligases predicted by analysis using the program Pfam.

Figure 9. Quality of the electron density map from the crystal structure of T. acidophilum LplA.

A β-strand with the sequence KLWHAA (residues 158–163) is illustrated.