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Structural ordering of disordered ligand-binding loops of biotin protein ligase into active conformations as a consequence of dehydration.

Gupta V, Gupta RK, Khare G, Salunke DM, Surolia A, Tyagi AK - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Delhi, New Delhi, India.

ABSTRACT
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.

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Dehydration induced structural ordering of L4 and L8 loops corresponds to active conformations.Superimposition of cartoon representation of subunit A of dhMtb-BirA (orange) onto EcBirA (2ewn, blue) and PhBirA (1wqw, magenta). Dehydration induced structural appearance of L4 and L8 loops in dhMtb-BirA coincides with the active conformation of these loops on ligand-binding.
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pone-0009222-g004: Dehydration induced structural ordering of L4 and L8 loops corresponds to active conformations.Superimposition of cartoon representation of subunit A of dhMtb-BirA (orange) onto EcBirA (2ewn, blue) and PhBirA (1wqw, magenta). Dehydration induced structural appearance of L4 and L8 loops in dhMtb-BirA coincides with the active conformation of these loops on ligand-binding.

Mentions: The asymmetric unit of dehydrated crystal form containing two molecules is more compact and densely packed with a low solvent content of 28% compared to the hydrated crystal forms. Analysis of the two crystal forms shows the core regions to be identical, but some significant conformational plasticity is clearly visible among the loops (Figure 2b) leading to a relatively higher rmsd of 2.03 Å for 454 equivalent Cα positions between 2cgh and dhMtb-BirA. Large shift of ∼3.5 Å in the flexible loop L6 as well as in the β8-L11-β9 region is an apparent consequence of crystal dehydration. Additionally, the most interesting feature is the appearance of two missing loop regions L4 and L8 and 5 N-terminal residues in subunit A of the dhMtb-BirA (Figure 2b). The two missing loops L4 and L8 were built following the visible electron density at half and full occupancy, respectively. The electron density for loop L8 leaves little doubt as to the position of the atoms (Figure 2c) and though electron density for loop L4 is not complete, apex of this loop constituting residues R69 and H70 has a well-defined density (Figure 2c). Although L4 displays broken electron density associated with it, the original weak positive density, without any model built into it, improved markedly after loop building and did not exhibit any negative density, increasing our confidence in the modeled loop. Furthermore, it is interesting that position of these density guided built loops (subunit A) turns out to be very close to the ligand bound active loops (Figure 4) reported in EcBirA (2ewn) and PhBirA (1wqw) and hence the subunit A of dhMtb-BirA corresponds to the active form of the enzyme.


Structural ordering of disordered ligand-binding loops of biotin protein ligase into active conformations as a consequence of dehydration.

Gupta V, Gupta RK, Khare G, Salunke DM, Surolia A, Tyagi AK - PLoS ONE (2010)

Dehydration induced structural ordering of L4 and L8 loops corresponds to active conformations.Superimposition of cartoon representation of subunit A of dhMtb-BirA (orange) onto EcBirA (2ewn, blue) and PhBirA (1wqw, magenta). Dehydration induced structural appearance of L4 and L8 loops in dhMtb-BirA coincides with the active conformation of these loops on ligand-binding.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821413&req=5

pone-0009222-g004: Dehydration induced structural ordering of L4 and L8 loops corresponds to active conformations.Superimposition of cartoon representation of subunit A of dhMtb-BirA (orange) onto EcBirA (2ewn, blue) and PhBirA (1wqw, magenta). Dehydration induced structural appearance of L4 and L8 loops in dhMtb-BirA coincides with the active conformation of these loops on ligand-binding.
Mentions: The asymmetric unit of dehydrated crystal form containing two molecules is more compact and densely packed with a low solvent content of 28% compared to the hydrated crystal forms. Analysis of the two crystal forms shows the core regions to be identical, but some significant conformational plasticity is clearly visible among the loops (Figure 2b) leading to a relatively higher rmsd of 2.03 Å for 454 equivalent Cα positions between 2cgh and dhMtb-BirA. Large shift of ∼3.5 Å in the flexible loop L6 as well as in the β8-L11-β9 region is an apparent consequence of crystal dehydration. Additionally, the most interesting feature is the appearance of two missing loop regions L4 and L8 and 5 N-terminal residues in subunit A of the dhMtb-BirA (Figure 2b). The two missing loops L4 and L8 were built following the visible electron density at half and full occupancy, respectively. The electron density for loop L8 leaves little doubt as to the position of the atoms (Figure 2c) and though electron density for loop L4 is not complete, apex of this loop constituting residues R69 and H70 has a well-defined density (Figure 2c). Although L4 displays broken electron density associated with it, the original weak positive density, without any model built into it, improved markedly after loop building and did not exhibit any negative density, increasing our confidence in the modeled loop. Furthermore, it is interesting that position of these density guided built loops (subunit A) turns out to be very close to the ligand bound active loops (Figure 4) reported in EcBirA (2ewn) and PhBirA (1wqw) and hence the subunit A of dhMtb-BirA corresponds to the active form of the enzyme.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Delhi, New Delhi, India.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus