Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high
fatty acid content that plays a crucial role in its pathogenesis.
Acetyl Coenzyme A Carboxylase (ACC), an important
enzyme that catalyzes the first reaction of
fatty acid biosynthesis, is biotinylated by
biotin acetyl-CoA carboxylase ligase (BirA). The
ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon
ligand-binding. Here, we report that
dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to
paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible
ligand-binding loops L4 and L8 in apo BirA. In addition, crystal
dehydration resulted in a shift of approximately 3.5 A in the flexible loop L6, a
proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on
dehydration emulates the action responsible for the complex formation with its
protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the
ligand-bound and
ligand-free states of the
protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward
ATP and
biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the
protein in the absence of any
ligand-bound structure, but also demonstrates the merit of
dehydration of crystals as an inimitable technique to have a glance at
proteins in action.