Limits...
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.

Show MeSH

Related in: MedlinePlus

Overall simulations results for E. coli DHDPS tetramer and dimer.(A) Cα RMSDs over the course of the simulations, for dimers from tet-1 & tet-2 (shades of grey), dim-A (blue), dim-B (light blue); (B) Cartoon representation of monomer-monomer reorientation during simulation of dimers. The relative rotation of monomers is represented by dotted lines and an arrow. Cartoons are shown for extreme conformations taken from dim-B (light-blue at 70 ns, blue at 433 ns), and mrsa-1 (green at 430 ns). Cα RMSD between extreme conformations are: 4.0 Å for the E.coli and 3.8 Å for the MRSA dimers. (C) Cα RMSD values for monomers from tet-1 & tet-2 (shades of grey), dim-A & dim-B (shades of blue); (D) Angles of rotation corresponding to monomer rearrangement. Only tet1-A (black), dim-A (blue) and dim-B (light blue) are represented for clarity, the thick lines represent the spline fit of the values.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3369909&req=5

pcbi-1002537-g002: Overall simulations results for E. coli DHDPS tetramer and dimer.(A) Cα RMSDs over the course of the simulations, for dimers from tet-1 & tet-2 (shades of grey), dim-A (blue), dim-B (light blue); (B) Cartoon representation of monomer-monomer reorientation during simulation of dimers. The relative rotation of monomers is represented by dotted lines and an arrow. Cartoons are shown for extreme conformations taken from dim-B (light-blue at 70 ns, blue at 433 ns), and mrsa-1 (green at 430 ns). Cα RMSD between extreme conformations are: 4.0 Å for the E.coli and 3.8 Å for the MRSA dimers. (C) Cα RMSD values for monomers from tet-1 & tet-2 (shades of grey), dim-A & dim-B (shades of blue); (D) Angles of rotation corresponding to monomer rearrangement. Only tet1-A (black), dim-A (blue) and dim-B (light blue) are represented for clarity, the thick lines represent the spline fit of the values.

Mentions: Both tetramer simulations consistently exhibited steady dynamics and reached an RMSD plateau from 80 ns until the end of the simulations with an RMSD = 1.5 Å, only slightly deviating from the crystal structure conformation (Figure 2A, grey lines; Video S1). In comparison the L197Y mutant dimer simulation (dim-A) showed a strikingly different behaviour (Figure 2A, blue; Video S2). While the Cα−RMSD curve remained close to the tetramer simulations for the first 150 ns, it increased to reach a RMSD plateau at ∼3.1 Å for the last 200 ns of simulation. Closer examination revealed that the increase in RMSD is largely a result of the 15 degrees relative re-orientation of monomers within the dimer (Figure 2B). RMSDs of Cα atoms within individual monomers in dim-A remained low throughout the simulations (mean RMSD ∼1.5 Å, Figure 2C), comparable to the steady RMSDs observed in all monomers simulations of tet-1 and tet-2 (mean RMSD = 1.1 Å). This indicates that the monomers experience relatively little structural deviation from their crystal conformation individually in dim-A, but undergo significant rigid-body motion, relative to each other, within the dimer. The angle of rotation of the monomers for the dim-A simulation is represented in Figure 2D (blue).


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)

Overall simulations results for E. coli DHDPS tetramer and dimer.(A) Cα RMSDs over the course of the simulations, for dimers from tet-1 & tet-2 (shades of grey), dim-A (blue), dim-B (light blue); (B) Cartoon representation of monomer-monomer reorientation during simulation of dimers. The relative rotation of monomers is represented by dotted lines and an arrow. Cartoons are shown for extreme conformations taken from dim-B (light-blue at 70 ns, blue at 433 ns), and mrsa-1 (green at 430 ns). Cα RMSD between extreme conformations are: 4.0 Å for the E.coli and 3.8 Å for the MRSA dimers. (C) Cα RMSD values for monomers from tet-1 & tet-2 (shades of grey), dim-A & dim-B (shades of blue); (D) Angles of rotation corresponding to monomer rearrangement. Only tet1-A (black), dim-A (blue) and dim-B (light blue) are represented for clarity, the thick lines represent the spline fit of the values.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002537-g002: Overall simulations results for E. coli DHDPS tetramer and dimer.(A) Cα RMSDs over the course of the simulations, for dimers from tet-1 & tet-2 (shades of grey), dim-A (blue), dim-B (light blue); (B) Cartoon representation of monomer-monomer reorientation during simulation of dimers. The relative rotation of monomers is represented by dotted lines and an arrow. Cartoons are shown for extreme conformations taken from dim-B (light-blue at 70 ns, blue at 433 ns), and mrsa-1 (green at 430 ns). Cα RMSD between extreme conformations are: 4.0 Å for the E.coli and 3.8 Å for the MRSA dimers. (C) Cα RMSD values for monomers from tet-1 & tet-2 (shades of grey), dim-A & dim-B (shades of blue); (D) Angles of rotation corresponding to monomer rearrangement. Only tet1-A (black), dim-A (blue) and dim-B (light blue) are represented for clarity, the thick lines represent the spline fit of the values.
Mentions: Both tetramer simulations consistently exhibited steady dynamics and reached an RMSD plateau from 80 ns until the end of the simulations with an RMSD = 1.5 Å, only slightly deviating from the crystal structure conformation (Figure 2A, grey lines; Video S1). In comparison the L197Y mutant dimer simulation (dim-A) showed a strikingly different behaviour (Figure 2A, blue; Video S2). While the Cα−RMSD curve remained close to the tetramer simulations for the first 150 ns, it increased to reach a RMSD plateau at ∼3.1 Å for the last 200 ns of simulation. Closer examination revealed that the increase in RMSD is largely a result of the 15 degrees relative re-orientation of monomers within the dimer (Figure 2B). RMSDs of Cα atoms within individual monomers in dim-A remained low throughout the simulations (mean RMSD ∼1.5 Å, Figure 2C), comparable to the steady RMSDs observed in all monomers simulations of tet-1 and tet-2 (mean RMSD = 1.1 Å). This indicates that the monomers experience relatively little structural deviation from their crystal conformation individually in dim-A, but undergo significant rigid-body motion, relative to each other, within the dimer. The angle of rotation of the monomers for the dim-A simulation is represented in Figure 2D (blue).

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