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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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The asymmetric dimer.(a) Superposition of cartoon representations of dhMtb-BirA subunit A (orange) and subunit B (green) exhibiting the structural differences in two subunits. Seven N-terminal residues and loop L4 are disordered in subunit B and have not been built. Maximum conformational differences in the two subunits are displayed in loop L8 with 14 Å shift measured at the apex of the loop. Inset shows the sigma weighed 2 Fo–Fc electron density map (gray mesh) contoured at 0.7σ around the L8 loop in subunit B of dhMtb-BirA (represented as sticks in atom type colors). (b) The dimeric molecule in the asymmetric unit of dhMtb-BirA (green) and hMtb-BirA (red) are shown after superposition of subunit A. The arrow indicates an anticlockwise rotation (7°) of the twofold axis that relates to the two monomers (indicated in the same color) required for subunit B of dhMtb-BirA to superpose on the corresponding subunit of hMtb-BirA.
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pone-0009222-g006: The asymmetric dimer.(a) Superposition of cartoon representations of dhMtb-BirA subunit A (orange) and subunit B (green) exhibiting the structural differences in two subunits. Seven N-terminal residues and loop L4 are disordered in subunit B and have not been built. Maximum conformational differences in the two subunits are displayed in loop L8 with 14 Å shift measured at the apex of the loop. Inset shows the sigma weighed 2 Fo–Fc electron density map (gray mesh) contoured at 0.7σ around the L8 loop in subunit B of dhMtb-BirA (represented as sticks in atom type colors). (b) The dimeric molecule in the asymmetric unit of dhMtb-BirA (green) and hMtb-BirA (red) are shown after superposition of subunit A. The arrow indicates an anticlockwise rotation (7°) of the twofold axis that relates to the two monomers (indicated in the same color) required for subunit B of dhMtb-BirA to superpose on the corresponding subunit of hMtb-BirA.

Mentions: The two subunits of dhMtb-BirA have structural differences in their loops and defy the expected two fold symmetry resulting in an asymmetric dimer where, subunit A can be considered a representation of the active ligand bound conformation of BirA and subunit B is more an icon of apo BirA with disordered loops. The rmsds between the two subunits of dhMtb-BirA are 0.887 Å for 198 Cα equivalent atoms as opposed to the rmsds of 0.578 Å for 208 Cα pairs of the hMtb-BirA subunits. The maximum deviation of ∼14 Å appears to be in L8 loop, which is adenosine binding loop (Figure 6a) and may correspond to the conformational shift this loop undergoes upon biotin or biotinyl-5′-AMP binding. A noncrystallographic dyad exists between the two subunits in both hMtb-BirA and dhMtb-BirA structures. Although the mode of dimerization is same, small differences are observed in the mutual orientation of the subunits in the dimer after superposing the A subunits of two structures (Figure 6b). The angle of rotation necessary for superposing the B subunits turns out to be nearly 7°.


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)

The asymmetric dimer.(a) Superposition of cartoon representations of dhMtb-BirA subunit A (orange) and subunit B (green) exhibiting the structural differences in two subunits. Seven N-terminal residues and loop L4 are disordered in subunit B and have not been built. Maximum conformational differences in the two subunits are displayed in loop L8 with 14 Å shift measured at the apex of the loop. Inset shows the sigma weighed 2 Fo–Fc electron density map (gray mesh) contoured at 0.7σ around the L8 loop in subunit B of dhMtb-BirA (represented as sticks in atom type colors). (b) The dimeric molecule in the asymmetric unit of dhMtb-BirA (green) and hMtb-BirA (red) are shown after superposition of subunit A. The arrow indicates an anticlockwise rotation (7°) of the twofold axis that relates to the two monomers (indicated in the same color) required for subunit B of dhMtb-BirA to superpose on the corresponding subunit of hMtb-BirA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009222-g006: The asymmetric dimer.(a) Superposition of cartoon representations of dhMtb-BirA subunit A (orange) and subunit B (green) exhibiting the structural differences in two subunits. Seven N-terminal residues and loop L4 are disordered in subunit B and have not been built. Maximum conformational differences in the two subunits are displayed in loop L8 with 14 Å shift measured at the apex of the loop. Inset shows the sigma weighed 2 Fo–Fc electron density map (gray mesh) contoured at 0.7σ around the L8 loop in subunit B of dhMtb-BirA (represented as sticks in atom type colors). (b) The dimeric molecule in the asymmetric unit of dhMtb-BirA (green) and hMtb-BirA (red) are shown after superposition of subunit A. The arrow indicates an anticlockwise rotation (7°) of the twofold axis that relates to the two monomers (indicated in the same color) required for subunit B of dhMtb-BirA to superpose on the corresponding subunit of hMtb-BirA.
Mentions: The two subunits of dhMtb-BirA have structural differences in their loops and defy the expected two fold symmetry resulting in an asymmetric dimer where, subunit A can be considered a representation of the active ligand bound conformation of BirA and subunit B is more an icon of apo BirA with disordered loops. The rmsds between the two subunits of dhMtb-BirA are 0.887 Å for 198 Cα equivalent atoms as opposed to the rmsds of 0.578 Å for 208 Cα pairs of the hMtb-BirA subunits. The maximum deviation of ∼14 Å appears to be in L8 loop, which is adenosine binding loop (Figure 6a) and may correspond to the conformational shift this loop undergoes upon biotin or biotinyl-5′-AMP binding. A noncrystallographic dyad exists between the two subunits in both hMtb-BirA and dhMtb-BirA structures. Although the mode of dimerization is same, small differences are observed in the mutual orientation of the subunits in the dimer after superposing the A subunits of two structures (Figure 6b). The angle of rotation necessary for superposing the B subunits turns out to be nearly 7°.

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