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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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Related in: MedlinePlus

Loop sequence diversity among BirAs.Loops and turns in Mtb-BirA and equivalent sequences in homologues (the first and the last residue numbers are indicated before and after each loop) are represented. The important residues identified by mutational studies in EcBirA are marked in red.
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pone-0009222-g003: Loop sequence diversity among BirAs.Loops and turns in Mtb-BirA and equivalent sequences in homologues (the first and the last residue numbers are indicated before and after each loop) are represented. The important residues identified by mutational studies in EcBirA are marked in red.

Mentions: Structures of dehydrated (dhMtb-BirA) and hydrated (hMtb-BirA) Mtb-BirA, were determined at 2.69 Å and 2.8 Å, respectively, using crystals grown under similar conditions. The hydrated structure contains 44% solvent content similar to that of Mtb-BirA structure (45.4%) solved at a resolution of 1.8 Å (PDB entry: 2cgh). The asymmetric unit for both hydrated crystal forms (2cgh and hMtb-BirA) includes two monomers in the asymmetric unit. Both the crystal structures are quite similar as indicated by the root-mean-square difference (rmsd) of 0.69 Å for 480 equivalent Cα atom pairs. The backbone structures of subunits A as superimposed with RAPIDO do not display any significant difference between the two structures either (Figure 2a). The Mtb-BirA molecule is composed of two domains; N-terminal domain 1 comprises of 7 β-strands (β1: 29–32, β2: 55–59, β3: 82–88, β4: 124–126, β5: 131–133, β6: 137–147 and β7: 150–158) and 6 α-helices (α1: 13–20, α2: 39–48, α3: 95–114, α4: 173–176, α5: 183–203, α6: 206–215), while C-terminal domain 2 is a SH3 domain with 5 strands forming an antiparallel β-sheet (β8: 220–227, β9: 231–240, β10: 246–250, β11: 253–257 and β12: 261–264). The major loops (Figure 3) in domain 1 are: L1 (21–28), L2 (33–38), L3 (49–54), L4 (60–81), L5 (89–94), L6 (115–123), L7 (127–130), L8 (159–172) & L9 (177–182) and in domain 2 are: L11 (228–230), L12 (241–245), L13 (251–252) & L14 (258–260). The two domains are linked together with loop L10 (216–219) that connects helix α6 of domain 1 and β8 strand of domain 2. The 7 N-terminus residues and two loop regions between residues 65–76 (L4) and 162–169 (L8) are not there in both subunits of high (1.8 Å, 2cgh) and low-resolution structures (2.8 Å, hMtb-BirA). The disordered loops are undetectable in other BirA structures (1bia, 1wq7 and 3fjp) as well and are associated with the conformational changes upon biotinyl-5′-AMP binding [12]–[14].


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)

Loop sequence diversity among BirAs.Loops and turns in Mtb-BirA and equivalent sequences in homologues (the first and the last residue numbers are indicated before and after each loop) are represented. The important residues identified by mutational studies in EcBirA are marked in red.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009222-g003: Loop sequence diversity among BirAs.Loops and turns in Mtb-BirA and equivalent sequences in homologues (the first and the last residue numbers are indicated before and after each loop) are represented. The important residues identified by mutational studies in EcBirA are marked in red.
Mentions: Structures of dehydrated (dhMtb-BirA) and hydrated (hMtb-BirA) Mtb-BirA, were determined at 2.69 Å and 2.8 Å, respectively, using crystals grown under similar conditions. The hydrated structure contains 44% solvent content similar to that of Mtb-BirA structure (45.4%) solved at a resolution of 1.8 Å (PDB entry: 2cgh). The asymmetric unit for both hydrated crystal forms (2cgh and hMtb-BirA) includes two monomers in the asymmetric unit. Both the crystal structures are quite similar as indicated by the root-mean-square difference (rmsd) of 0.69 Å for 480 equivalent Cα atom pairs. The backbone structures of subunits A as superimposed with RAPIDO do not display any significant difference between the two structures either (Figure 2a). The Mtb-BirA molecule is composed of two domains; N-terminal domain 1 comprises of 7 β-strands (β1: 29–32, β2: 55–59, β3: 82–88, β4: 124–126, β5: 131–133, β6: 137–147 and β7: 150–158) and 6 α-helices (α1: 13–20, α2: 39–48, α3: 95–114, α4: 173–176, α5: 183–203, α6: 206–215), while C-terminal domain 2 is a SH3 domain with 5 strands forming an antiparallel β-sheet (β8: 220–227, β9: 231–240, β10: 246–250, β11: 253–257 and β12: 261–264). The major loops (Figure 3) in domain 1 are: L1 (21–28), L2 (33–38), L3 (49–54), L4 (60–81), L5 (89–94), L6 (115–123), L7 (127–130), L8 (159–172) & L9 (177–182) and in domain 2 are: L11 (228–230), L12 (241–245), L13 (251–252) & L14 (258–260). The two domains are linked together with loop L10 (216–219) that connects helix α6 of domain 1 and β8 strand of domain 2. The 7 N-terminus residues and two loop regions between residues 65–76 (L4) and 162–169 (L8) are not there in both subunits of high (1.8 Å, 2cgh) and low-resolution structures (2.8 Å, hMtb-BirA). The disordered loops are undetectable in other BirA structures (1bia, 1wq7 and 3fjp) as well and are associated with the conformational changes upon biotinyl-5′-AMP binding [12]–[14].

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