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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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BirA-BCCP complex.(a) Structural superposition of cartoon representations of subunits A of apo (yellow) and BCCP complexed (magenta) PhBirA illustrating the open/close movement (marked by an arrow) of L14 loop to regulate the entry/exit of BCCP. (b) Similar superposition of hMtb-BirA (green) and dhMtb-BirA (orange) representing apo and active forms, respectively, demonstrate no such movement in L14 loop. Contrary to the movement of C-terminal loops in PhBirA, the loop L11 in Mtb-BirA moves inwards (marked by an arrow) on BCCP binding. BCCP molecule in both figures is shown as blue cartoon.
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pone-0009222-g008: BirA-BCCP complex.(a) Structural superposition of cartoon representations of subunits A of apo (yellow) and BCCP complexed (magenta) PhBirA illustrating the open/close movement (marked by an arrow) of L14 loop to regulate the entry/exit of BCCP. (b) Similar superposition of hMtb-BirA (green) and dhMtb-BirA (orange) representing apo and active forms, respectively, demonstrate no such movement in L14 loop. Contrary to the movement of C-terminal loops in PhBirA, the loop L11 in Mtb-BirA moves inwards (marked by an arrow) on BCCP binding. BCCP molecule in both figures is shown as blue cartoon.

Mentions: Plasticity around the active site of PhBirA has been seen to assist in complex formation with its substrate BCCP [13]. In addition, superposition of apo and holo PhBirA structures exhibits maximum variations in the C-terminal domain, especially in L11 and L14 loops (Figure 8a). It has been proposed that the loop L14 involving the residue Y227 (P. horikoshii sequence in Figure 3) undergoes an open/close motion to regulate the movements of ligands. In the active complex, L11 and L14 loops shift outwards to place BCCP at the active site. However, the docking of modeled BCCP domain of Mtb ACCA3 in the hMtb-BirA and dhMtb-BirA, representing free and ligand bound states of Mtb-BirA, respectively, reveals contrary movements of these loops (Figure 8b). The loop L14 in Mtb structure is only three residues long and is devoid of the tyrosine residue Y227 (Figure 3). Moreover, the absence of any significant structural change in this very short loop negates its possible involvement during substrate binding. But the closing in of L11 loop in the Mtb-BirA BCCP complex put forward the notion that the ligand placement role of L14 in phBirA is carried out by L11 in Mtb. Interestingly, this loop does not appear to be involved in any gated mechanism for the entry/exit of BCCP substrate thus providing a constitutive access to the ligand. This Mtb specific behavior of C-terminal domain justifies the need for accommodating different BCCP domains [32] and biotinylation turnover necessary for the biosynthesis of unique fatty acids in Mtb under varying environmental conditions.


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)

BirA-BCCP complex.(a) Structural superposition of cartoon representations of subunits A of apo (yellow) and BCCP complexed (magenta) PhBirA illustrating the open/close movement (marked by an arrow) of L14 loop to regulate the entry/exit of BCCP. (b) Similar superposition of hMtb-BirA (green) and dhMtb-BirA (orange) representing apo and active forms, respectively, demonstrate no such movement in L14 loop. Contrary to the movement of C-terminal loops in PhBirA, the loop L11 in Mtb-BirA moves inwards (marked by an arrow) on BCCP binding. BCCP molecule in both figures is shown as blue cartoon.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009222-g008: BirA-BCCP complex.(a) Structural superposition of cartoon representations of subunits A of apo (yellow) and BCCP complexed (magenta) PhBirA illustrating the open/close movement (marked by an arrow) of L14 loop to regulate the entry/exit of BCCP. (b) Similar superposition of hMtb-BirA (green) and dhMtb-BirA (orange) representing apo and active forms, respectively, demonstrate no such movement in L14 loop. Contrary to the movement of C-terminal loops in PhBirA, the loop L11 in Mtb-BirA moves inwards (marked by an arrow) on BCCP binding. BCCP molecule in both figures is shown as blue cartoon.
Mentions: Plasticity around the active site of PhBirA has been seen to assist in complex formation with its substrate BCCP [13]. In addition, superposition of apo and holo PhBirA structures exhibits maximum variations in the C-terminal domain, especially in L11 and L14 loops (Figure 8a). It has been proposed that the loop L14 involving the residue Y227 (P. horikoshii sequence in Figure 3) undergoes an open/close motion to regulate the movements of ligands. In the active complex, L11 and L14 loops shift outwards to place BCCP at the active site. However, the docking of modeled BCCP domain of Mtb ACCA3 in the hMtb-BirA and dhMtb-BirA, representing free and ligand bound states of Mtb-BirA, respectively, reveals contrary movements of these loops (Figure 8b). The loop L14 in Mtb structure is only three residues long and is devoid of the tyrosine residue Y227 (Figure 3). Moreover, the absence of any significant structural change in this very short loop negates its possible involvement during substrate binding. But the closing in of L11 loop in the Mtb-BirA BCCP complex put forward the notion that the ligand placement role of L14 in phBirA is carried out by L11 in Mtb. Interestingly, this loop does not appear to be involved in any gated mechanism for the entry/exit of BCCP substrate thus providing a constitutive access to the ligand. This Mtb specific behavior of C-terminal domain justifies the need for accommodating different BCCP domains [32] and biotinylation turnover necessary for the biosynthesis of unique fatty acids in Mtb under varying environmental conditions.

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