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Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

Reboul CF, Porebski BT, Griffin MD, Dobson RC, Perugini MA, Gerrard JA, Buckle AM - PLoS Comput. Biol. (2012)

Bottom Line: DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface.Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity.These reveal a striking contrast between the dynamics of tetrameric and dimeric forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

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Snapshots of active site residues taken from: (A) E. coli tetramer (tet-1), (B) E. coli mutant dimer (dim-A), and (C) MRSA simulations (mrsa-1).Y107 (E. coli)/109 (MRSA) is highlighted in purple (A), blue (B) or pale green (C). Snaphots are taken every 100 ns from each trajectory.
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pcbi-1002537-g004: Snapshots of active site residues taken from: (A) E. coli tetramer (tet-1), (B) E. coli mutant dimer (dim-A), and (C) MRSA simulations (mrsa-1).Y107 (E. coli)/109 (MRSA) is highlighted in purple (A), blue (B) or pale green (C). Snaphots are taken every 100 ns from each trajectory.

Mentions: To estimate the extent of the active site deformation we calculated the RMSD values (heavy-atoms only) over all the simulations for the eight active residues (T44, Y106, Y133, R138, K161, G186, I203, and Y107 contributed by the adjacent monomer; Figure 1D). Active site residues in the tetramer simulations fluctuate within an RMSD range of 0.8–1.8 Å, with a mean of 1.0 Å, and are relatively stable in their conformation throughout the last 400 ns of the simulations (Figure 3A, grey lines; Figure 4A; Video S3). Conversely, the positions of active site residues in the dimer deviate from the crystal conformation to a much larger degree, with RMSD values varying from an initial 1.0 Å up to 2.8 Å (dim-A) and 3.5 Å (dim-B) towards the end of the simulations (Figure 3A, blue lines; Figure 4B; Video S4). Even though the residues in the dim-A and dim-B active sites show differences in their conformations, they both consistently deviate from the wild-type positions with RMSD values greater than 2 Å over the last 150 ns of the simulations. Our simulations demonstrate that the active sites show more deformation in dimers than in tetramers, where residues show relatively small deviations from their crystal conformation (Figure 4A,B). To estimate potential flexibility in the 8 amino acids composing the active site we calculated the root mean square fluctuations (RMSFs) for the tetramer and dimer simulations (Figure 3B). The results clearly show a general flexibility increase in the dimer active site compared to the tetramer. While the tetramer active site residues display individually low flexibility (RMSF range = 0.4–0.9 Å; Figure 3B and 4A; Video S3), dimer active site residues appear considerably more flexible (RMSF range = 0.6–2.4 Å; Figures 3B and 4B; Video S4). Interestingly, the catalytic residues T44 and Y107 as well as Y106 and R138 contribute most to the increased flexibility within the dimer active site. The remaining residues (Y133, K161, G186, I203) are also more flexible in the dimer compared to the tetramer, although they fluctuate somewhat less (RMSF values<1.0 Å).


Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

Reboul CF, Porebski BT, Griffin MD, Dobson RC, Perugini MA, Gerrard JA, Buckle AM - PLoS Comput. Biol. (2012)

Snapshots of active site residues taken from: (A) E. coli tetramer (tet-1), (B) E. coli mutant dimer (dim-A), and (C) MRSA simulations (mrsa-1).Y107 (E. coli)/109 (MRSA) is highlighted in purple (A), blue (B) or pale green (C). Snaphots are taken every 100 ns from each trajectory.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369909&req=5

pcbi-1002537-g004: Snapshots of active site residues taken from: (A) E. coli tetramer (tet-1), (B) E. coli mutant dimer (dim-A), and (C) MRSA simulations (mrsa-1).Y107 (E. coli)/109 (MRSA) is highlighted in purple (A), blue (B) or pale green (C). Snaphots are taken every 100 ns from each trajectory.
Mentions: To estimate the extent of the active site deformation we calculated the RMSD values (heavy-atoms only) over all the simulations for the eight active residues (T44, Y106, Y133, R138, K161, G186, I203, and Y107 contributed by the adjacent monomer; Figure 1D). Active site residues in the tetramer simulations fluctuate within an RMSD range of 0.8–1.8 Å, with a mean of 1.0 Å, and are relatively stable in their conformation throughout the last 400 ns of the simulations (Figure 3A, grey lines; Figure 4A; Video S3). Conversely, the positions of active site residues in the dimer deviate from the crystal conformation to a much larger degree, with RMSD values varying from an initial 1.0 Å up to 2.8 Å (dim-A) and 3.5 Å (dim-B) towards the end of the simulations (Figure 3A, blue lines; Figure 4B; Video S4). Even though the residues in the dim-A and dim-B active sites show differences in their conformations, they both consistently deviate from the wild-type positions with RMSD values greater than 2 Å over the last 150 ns of the simulations. Our simulations demonstrate that the active sites show more deformation in dimers than in tetramers, where residues show relatively small deviations from their crystal conformation (Figure 4A,B). To estimate potential flexibility in the 8 amino acids composing the active site we calculated the root mean square fluctuations (RMSFs) for the tetramer and dimer simulations (Figure 3B). The results clearly show a general flexibility increase in the dimer active site compared to the tetramer. While the tetramer active site residues display individually low flexibility (RMSF range = 0.4–0.9 Å; Figure 3B and 4A; Video S3), dimer active site residues appear considerably more flexible (RMSF range = 0.6–2.4 Å; Figures 3B and 4B; Video S4). Interestingly, the catalytic residues T44 and Y107 as well as Y106 and R138 contribute most to the increased flexibility within the dimer active site. The remaining residues (Y133, K161, G186, I203) are also more flexible in the dimer compared to the tetramer, although they fluctuate somewhat less (RMSF values<1.0 Å).

Bottom Line: DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface.Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity.These reveal a striking contrast between the dynamics of tetrameric and dimeric forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

Show MeSH
Related in: MedlinePlus