The wing of a winged helix-turn-helix transcription factor organizes the active site of BirA, a bifunctional repressor/ligase

Vandana Chakravartty, John E. Cronan

Research output: Contribution to journalArticlepeer-review

Abstract

The BirA biotin protein ligase of Escherichia coli belongs to the winged helix-turn-helix (wHTH) family of transcriptional regulators. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity. We addressed the structural and functional role of the wing of the wHTH motif in both BirA functions. A panel of N-terminal deletion mutant proteins including a discrete deletion of the wing motif were unable to bind DNA. However, all the N-terminal deletion mutants weakly complemented growth of a ΔbirA strain at low biotin concentrations, indicating compromised ligase activity. A wing domain chimera was constructed by replacing the BirA wing with the nearly isosteric wing of the E. coli OmpR transcription factor. Although this chimera BirA was defective in operator binding, it was much more efficient in complementation of a ΔbirA strain than was the wing-less protein. The enzymatic activities of the wing deletion and chimera proteins in the in vitro synthesis of biotinoyl-5′-AMP differed greatly. The wing deletion BirA accumulated an off pathway compound, ADP, whereas the chimera protein did not. Finally, we report that a single residue alteration in the wing bypasses the deleterious effects caused by mutations in the biotin-binding loop of the ligase active site. We believe that the role of the wing in the BirA enzymatic reaction is to orient the active site and thereby protect biotinoyl-5′-AMP from attack by solvent. This is the first evidence that the wing domain of a wHTH protein can play an important role in enzymatic activity.

Original languageEnglish (US)
Pages (from-to)36029-36039
Number of pages11
JournalJournal of Biological Chemistry
Volume288
Issue number50
DOIs
StatePublished - Dec 13 2013

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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