Abstract
Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL.BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5'-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5'-AMP, thereby providing the complex crystals. The four crystallographically independent BPL.BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction.
Highlights
MAY 23, 2008 VOLUME 283 NUMBER 21 enzymes is catalyzed by the biotin protein ligase (BPL)2 in two steps (Reactions 1 and 2)
BPL activates biotin at the expense of ATP to the reaction intermediate biotinyl-5Ј-AMP in which the carboxyl group of inert biotin is activated by the addition of an adenylate group
The biotin moiety of biotinyl-5Ј-AMP is transferred to the ⑀-amino group of a specific lysine residue of the target protein (e.g. the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase [3])
Summary
Protein Expression, Purification, Crystallization, and Data Collection—The expression and purification of PhBPL, PhBPL*, PhBPL**, and PhBCCP⌬N76 were performed as described elsewhere [15, 16]. Model Building and Refinement—The structures of the mutated and/or liganded forms of PhBPL were determined by the difference Fourier synthesis based on its wild-type structure. The structures of PhBCCP⌬N76 in two different crystal forms were determined by the molecular replacement with the program MOLREP [19], using coordinates of the C-terminal domain of E. coli BCCP (EcBCCP, PDB ID 1BDO) as a search model [20]. The structural superposition analysis was performed using the program LSQKAB [23] and a subsequent classification of structural changes according to the multiple superposition method [24].
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