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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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Xenon binding in StPurL.(A) StPurL structure is depicted in cartoon with N-terminal domain in green, linker domain in yellow, glutaminase domain in red, and FGAM synthetase domain with structural sub-domains 1 and 2 shown in blue and cyan colors respectively. The two active sites and the auxillary ADP are shown in sticks and Xenon atoms are depicted as orange spheres. In the side panel (B, C and D), mFo-DFc densitiy maps of the three Xenon atoms at 5.0 σ are shown in black mesh and residues forming the cavities around them are shown in sticks.
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pone-0077781-g001: Xenon binding in StPurL.(A) StPurL structure is depicted in cartoon with N-terminal domain in green, linker domain in yellow, glutaminase domain in red, and FGAM synthetase domain with structural sub-domains 1 and 2 shown in blue and cyan colors respectively. The two active sites and the auxillary ADP are shown in sticks and Xenon atoms are depicted as orange spheres. In the side panel (B, C and D), mFo-DFc densitiy maps of the three Xenon atoms at 5.0 σ are shown in black mesh and residues forming the cavities around them are shown in sticks.

Mentions: Diffraction data for StPurL-Xenon complex was collected at X12 beamline of European Molecular Biology Laboratory (EMBL)-Hamburg. 200 images were collected for each data set. In order to collect this data, crystals of native StPurL were grown by vapor diffusion as described previously [13] and subsequently they were exposed to xenon gas at 40 bar using a pressurization chamber from Hampton Research [23]. The crystals were cryo-cooled immediately after de pressurization and tested for X-ray diffraction at 100 K and the data collected. Multiple data sets were collected with crystals pressurized for different durations ranging from 30 sec to 5 min. The Auto-Rickshaw automated crystal structure-determination software [24] was used to confirm at the beamline that the appropriate xenon derivative data had been captured. The crystals pressurized for 1 min diffracted to a resolution of 2.18 Å for the R1263A mutant and 2.65 Å for the native. The larger size of the R1263A mutant crystals enabled collection of higher resolution data perhaps due to larger beam-size at the beamline. Data was collected using a MAR225 detector, 0.5° oscillation, 30 sec exposure time and 150 mm crystal to detector distance at a wavelength of 1.37 Å. Crystals of F209W mutant were also grown by vapor diffusion as described above and diffraction data was collected at BM14 beamline of European Synchrotron Radiation Facility (ESRF), Grenoble. The data for StPurL-Xenon and F209W mutant, like that of the native crystals were in the hexagonal space group P65 with unit cell dimensions a = 146.68 Å, c = 141.22 Å for the xenon complex (PDB ID 4L78) and a = 146.33 Å, c = 140.84 Å for F209W mutant (PDB ID 4LGY). The data were indexed, integrated in iMosflm and scaled using SCALA and CCP4 [25] suite of programs. The structures were determined by performing rigid body refinement against the published structure of StPurL (protein data bank (PDB) ID: IT3T) using the program Refmac5 in CCP4i [26] suite. The xenon bound sites were identified by constructing both an anomalous difference Fourier (Figure S2) and mFo-DFc maps (Figure 1). The initial models were subsequently refined by performing rounds of refinement using Refmac5 [27] followed by manual model building using the program Coot [28]. Cavity volumes for the xenon binding sites were calculated using the program Deepview Swiss-PdbViewer [29].


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)

Xenon binding in StPurL.(A) StPurL structure is depicted in cartoon with N-terminal domain in green, linker domain in yellow, glutaminase domain in red, and FGAM synthetase domain with structural sub-domains 1 and 2 shown in blue and cyan colors respectively. The two active sites and the auxillary ADP are shown in sticks and Xenon atoms are depicted as orange spheres. In the side panel (B, C and D), mFo-DFc densitiy maps of the three Xenon atoms at 5.0 σ are shown in black mesh and residues forming the cavities around them are shown in sticks.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815217&req=5

pone-0077781-g001: Xenon binding in StPurL.(A) StPurL structure is depicted in cartoon with N-terminal domain in green, linker domain in yellow, glutaminase domain in red, and FGAM synthetase domain with structural sub-domains 1 and 2 shown in blue and cyan colors respectively. The two active sites and the auxillary ADP are shown in sticks and Xenon atoms are depicted as orange spheres. In the side panel (B, C and D), mFo-DFc densitiy maps of the three Xenon atoms at 5.0 σ are shown in black mesh and residues forming the cavities around them are shown in sticks.
Mentions: Diffraction data for StPurL-Xenon complex was collected at X12 beamline of European Molecular Biology Laboratory (EMBL)-Hamburg. 200 images were collected for each data set. In order to collect this data, crystals of native StPurL were grown by vapor diffusion as described previously [13] and subsequently they were exposed to xenon gas at 40 bar using a pressurization chamber from Hampton Research [23]. The crystals were cryo-cooled immediately after de pressurization and tested for X-ray diffraction at 100 K and the data collected. Multiple data sets were collected with crystals pressurized for different durations ranging from 30 sec to 5 min. The Auto-Rickshaw automated crystal structure-determination software [24] was used to confirm at the beamline that the appropriate xenon derivative data had been captured. The crystals pressurized for 1 min diffracted to a resolution of 2.18 Å for the R1263A mutant and 2.65 Å for the native. The larger size of the R1263A mutant crystals enabled collection of higher resolution data perhaps due to larger beam-size at the beamline. Data was collected using a MAR225 detector, 0.5° oscillation, 30 sec exposure time and 150 mm crystal to detector distance at a wavelength of 1.37 Å. Crystals of F209W mutant were also grown by vapor diffusion as described above and diffraction data was collected at BM14 beamline of European Synchrotron Radiation Facility (ESRF), Grenoble. The data for StPurL-Xenon and F209W mutant, like that of the native crystals were in the hexagonal space group P65 with unit cell dimensions a = 146.68 Å, c = 141.22 Å for the xenon complex (PDB ID 4L78) and a = 146.33 Å, c = 140.84 Å for F209W mutant (PDB ID 4LGY). The data were indexed, integrated in iMosflm and scaled using SCALA and CCP4 [25] suite of programs. The structures were determined by performing rigid body refinement against the published structure of StPurL (protein data bank (PDB) ID: IT3T) using the program Refmac5 in CCP4i [26] suite. The xenon bound sites were identified by constructing both an anomalous difference Fourier (Figure S2) and mFo-DFc maps (Figure 1). The initial models were subsequently refined by performing rounds of refinement using Refmac5 [27] followed by manual model building using the program Coot [28]. Cavity volumes for the xenon binding sites were calculated using the program Deepview Swiss-PdbViewer [29].

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