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Importance of hydrophobic cavities in allosteric regulation of formylglycinamide synthetase: insight from xenon trapping and statistical coupling analysis.

Tanwar AS, Goyal VD, Choudhary D, Panjikar S, Anand R - PLoS ONE (2013)

Bottom Line: Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers.It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot.In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, India.

ABSTRACT
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

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Related in: MedlinePlus

Loop regions around cavity 2.(A) Glutaminase domain is shown in surface representation in purple color with active site residues, helix α27 and α30 highlighted in cartoon and stick representations. A2 domain of the FGAM synthetase is shown in pink cartoons with the long and short loops in green color. The structurally conserved long loop region of TmPurL is shown in yellow cartoon. ADP is shown in magenta sticks. The β-sheet regions of A1 domain involved in making the β-barrel core of the FGAM synthetase domain are shown in marine blue cartoon representation. Locations of the xenon atoms trapped in the structure are depicted as orange spheres. (B) View of the interface between the FGAM synthetase loop regions and the glutaminase domain showing various hydrogen bonding and van der waals interactions.
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pone-0077781-g007: Loop regions around cavity 2.(A) Glutaminase domain is shown in surface representation in purple color with active site residues, helix α27 and α30 highlighted in cartoon and stick representations. A2 domain of the FGAM synthetase is shown in pink cartoons with the long and short loops in green color. The structurally conserved long loop region of TmPurL is shown in yellow cartoon. ADP is shown in magenta sticks. The β-sheet regions of A1 domain involved in making the β-barrel core of the FGAM synthetase domain are shown in marine blue cartoon representation. Locations of the xenon atoms trapped in the structure are depicted as orange spheres. (B) View of the interface between the FGAM synthetase loop regions and the glutaminase domain showing various hydrogen bonding and van der waals interactions.

Mentions: As mentioned earlier, the Xe2 atom was trapped in a small cavity at the edge of the central β-barrel of the FGAM synthetase domain. The strands constituting the wall of the cavity were connected to two loops, a short and a long one that form a flap. Analysis of the long loop shows that it is buried into the protein and forms part of the interface between glutaminase and the FGAM synthetase domains via a conserved network of interactions (Figure 7A). A comparison of the available crystal structures of TmPurSSQL (PDB ID 3D54) with StPurL further revealed that TmPurL has retained a majority of this long interdomain loop region and that it shows good structural superposition with that of StPurL (Figure 7). The significance of this loop was additionally highlighted, more so because of the thermophilic nature of TmPurL several loops have been deleted in this protein to attain structural stability at higher temperatures, however, this interdomain loop is retained (Figure 7B). Furthermore, it was noticed that the N-terminal region of the long loop is in close contact with the auxiliary ADP binding site and residue Asp679 lying on this loop was in close contact of the second phosphate of the ADP.


Importance of hydrophobic cavities in allosteric regulation of formylglycinamide synthetase: insight from xenon trapping and statistical coupling analysis.

Tanwar AS, Goyal VD, Choudhary D, Panjikar S, Anand R - PLoS ONE (2013)

Loop regions around cavity 2.(A) Glutaminase domain is shown in surface representation in purple color with active site residues, helix α27 and α30 highlighted in cartoon and stick representations. A2 domain of the FGAM synthetase is shown in pink cartoons with the long and short loops in green color. The structurally conserved long loop region of TmPurL is shown in yellow cartoon. ADP is shown in magenta sticks. The β-sheet regions of A1 domain involved in making the β-barrel core of the FGAM synthetase domain are shown in marine blue cartoon representation. Locations of the xenon atoms trapped in the structure are depicted as orange spheres. (B) View of the interface between the FGAM synthetase loop regions and the glutaminase domain showing various hydrogen bonding and van der waals interactions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815217&req=5

pone-0077781-g007: Loop regions around cavity 2.(A) Glutaminase domain is shown in surface representation in purple color with active site residues, helix α27 and α30 highlighted in cartoon and stick representations. A2 domain of the FGAM synthetase is shown in pink cartoons with the long and short loops in green color. The structurally conserved long loop region of TmPurL is shown in yellow cartoon. ADP is shown in magenta sticks. The β-sheet regions of A1 domain involved in making the β-barrel core of the FGAM synthetase domain are shown in marine blue cartoon representation. Locations of the xenon atoms trapped in the structure are depicted as orange spheres. (B) View of the interface between the FGAM synthetase loop regions and the glutaminase domain showing various hydrogen bonding and van der waals interactions.
Mentions: As mentioned earlier, the Xe2 atom was trapped in a small cavity at the edge of the central β-barrel of the FGAM synthetase domain. The strands constituting the wall of the cavity were connected to two loops, a short and a long one that form a flap. Analysis of the long loop shows that it is buried into the protein and forms part of the interface between glutaminase and the FGAM synthetase domains via a conserved network of interactions (Figure 7A). A comparison of the available crystal structures of TmPurSSQL (PDB ID 3D54) with StPurL further revealed that TmPurL has retained a majority of this long interdomain loop region and that it shows good structural superposition with that of StPurL (Figure 7). The significance of this loop was additionally highlighted, more so because of the thermophilic nature of TmPurL several loops have been deleted in this protein to attain structural stability at higher temperatures, however, this interdomain loop is retained (Figure 7B). Furthermore, it was noticed that the N-terminal region of the long loop is in close contact with the auxiliary ADP binding site and residue Asp679 lying on this loop was in close contact of the second phosphate of the ADP.

Bottom Line: Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers.It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot.In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, India.

ABSTRACT
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

Show MeSH
Related in: MedlinePlus