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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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Characterization of StPurLMutants.(A) Secondary structure analysis: Wavelength scan using circular dichroism spectroscopy performed for all variants at the same molar concentration. (B) Stability: Thermal denaturation monitored using CD signal at 222 nm. (C) Activity (FGAM synthetase): Percentage activity of all mutants at 37°C with respect to that of native StPurL protein.
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pone-0077781-g003: Characterization of StPurLMutants.(A) Secondary structure analysis: Wavelength scan using circular dichroism spectroscopy performed for all variants at the same molar concentration. (B) Stability: Thermal denaturation monitored using CD signal at 222 nm. (C) Activity (FGAM synthetase): Percentage activity of all mutants at 37°C with respect to that of native StPurL protein.

Mentions: In order to get corroborating evidence for hypotheses about the importance of xenon binding regions mutagenesis studies were performed. These experiments were aimed at perturbing the xenon binding cavities so as to determine their effect on various aspects of protein function and stability. Candidates for site-directed mutagenesis were first screened in silico and mutations that showed possible rotamers that could adjust into the structure by filling the cavity without significant clashes with the neighboring residues were chosen. Mutation L1181Y seemed to be one of the most promising case in the in silico mutagenesis phase as it did not exhibit significant clashes but filled the cavity. However, the mutant was insoluble and no biophysical data could be collected suggesting that this residue was extremely deleterious to the structural integrity of the protein. As a result of the failure of L1181Y to yield soluble protein, less intrusive mutation L1181F and a bulkier mutation L1181W were subsequently made. Surprisingly the L1181F mutant had lower yield during expression and purification whereas the L1181W was comparable in yield to wild type. The circular dichroism (CD) signal (Figure 3A) also had a lower ellipticity than the wild-type protein for L1181F suggesting lower overall folding of the secondary structure elements for this mutant. Thermal denaturation (Figure 3B) showed that unlike the two step unfolding of the wild-type protein, this mutant had a one step unfolding profile with an unfolding temperature 15°C below the second unfolding transition of the wild-type protein indicating large destabilization. This mutation also caused a 60% loss in activity of the protein (Figure 3C). On the other hand, L1181W had secondary structure content and FGAM synthetase activity at par with the wild-type protein and exhibited almost no destabilization (Figure 3A, C). Thermal denaturation profile was also similar to the wild-type except that the second unfolding transition of the mutant was 5°C below that of the wild-type protein (Figure 3B). This mutagenesis results were intriguing as contrary to general belief, a bulkier residue with obvious clashes was accepted whereas phenylalanine residue with no obvious clash was unacceptable (Figure S8).


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)

Characterization of StPurLMutants.(A) Secondary structure analysis: Wavelength scan using circular dichroism spectroscopy performed for all variants at the same molar concentration. (B) Stability: Thermal denaturation monitored using CD signal at 222 nm. (C) Activity (FGAM synthetase): Percentage activity of all mutants at 37°C with respect to that of native StPurL protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0077781-g003: Characterization of StPurLMutants.(A) Secondary structure analysis: Wavelength scan using circular dichroism spectroscopy performed for all variants at the same molar concentration. (B) Stability: Thermal denaturation monitored using CD signal at 222 nm. (C) Activity (FGAM synthetase): Percentage activity of all mutants at 37°C with respect to that of native StPurL protein.
Mentions: In order to get corroborating evidence for hypotheses about the importance of xenon binding regions mutagenesis studies were performed. These experiments were aimed at perturbing the xenon binding cavities so as to determine their effect on various aspects of protein function and stability. Candidates for site-directed mutagenesis were first screened in silico and mutations that showed possible rotamers that could adjust into the structure by filling the cavity without significant clashes with the neighboring residues were chosen. Mutation L1181Y seemed to be one of the most promising case in the in silico mutagenesis phase as it did not exhibit significant clashes but filled the cavity. However, the mutant was insoluble and no biophysical data could be collected suggesting that this residue was extremely deleterious to the structural integrity of the protein. As a result of the failure of L1181Y to yield soluble protein, less intrusive mutation L1181F and a bulkier mutation L1181W were subsequently made. Surprisingly the L1181F mutant had lower yield during expression and purification whereas the L1181W was comparable in yield to wild type. The circular dichroism (CD) signal (Figure 3A) also had a lower ellipticity than the wild-type protein for L1181F suggesting lower overall folding of the secondary structure elements for this mutant. Thermal denaturation (Figure 3B) showed that unlike the two step unfolding of the wild-type protein, this mutant had a one step unfolding profile with an unfolding temperature 15°C below the second unfolding transition of the wild-type protein indicating large destabilization. This mutation also caused a 60% loss in activity of the protein (Figure 3C). On the other hand, L1181W had secondary structure content and FGAM synthetase activity at par with the wild-type protein and exhibited almost no destabilization (Figure 3A, C). Thermal denaturation profile was also similar to the wild-type except that the second unfolding transition of the mutant was 5°C below that of the wild-type protein (Figure 3B). This mutagenesis results were intriguing as contrary to general belief, a bulkier residue with obvious clashes was accepted whereas phenylalanine residue with no obvious clash was unacceptable (Figure S8).

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