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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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A detailed view of the tight-dimer interface in E. coli and MRSA DHDPS.Surfaces of both enzymes with the residues involved in the tight-dimer interface represented in light orange. Residues involved in hydrogen bonds are shown in red and in salt-bridges in yellow, as calculated by the PISA server (A) Dimer from E. coli wild-type tetramer (PDB ID: 1YXC); (B) MRSA wild-type dimer (PDB ID: 3DAQ).
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pcbi-1002537-g005: A detailed view of the tight-dimer interface in E. coli and MRSA DHDPS.Surfaces of both enzymes with the residues involved in the tight-dimer interface represented in light orange. Residues involved in hydrogen bonds are shown in red and in salt-bridges in yellow, as calculated by the PISA server (A) Dimer from E. coli wild-type tetramer (PDB ID: 1YXC); (B) MRSA wild-type dimer (PDB ID: 3DAQ).

Mentions: Our simulation data for E. coli DHDPS suggest that conformational fluctuations and flexibility at the active site is a primary cause of the dramatic decrease in enzymatic activity of dimers. The existence of a naturally occurring dimer from the bacterial pathogen MRSA that exhibits comparable activity to the E. coli tetramer is therefore intriguing [8]. Whereas the overall tertiary structures of MRSA and E. coli DHDPS are highly similar (RMSD = 0.9 Å; Figure 1B,C), with only minor reorientations of active site side-chains (Figure 1D), the nature of their tight-dimer interfaces differs (Figure 5). MRSA DHDPS possesses a relatively high number of hydrogen bonds at the tight-dimer interface and two electrostatic interactions that are absent in the E. coli structure, suggesting that it is perhaps less flexible that its E. coli counterpart [8]. We therefore performed two MD simulations of the MRSA DHDPS dimer in the absence of substrate and compared the results to the E. coli DHDPS simulations. The 1.45 Å resolution crystal structure of MRSA DHDPS [8] was used as the starting structure for two independent MD simulations of 0.5 µs each in length (denoted mrsa-1 and mrsa-2). Both simulations show a gradual increase in RMSD, which stabilise and reach a plateau at ∼3 Å at ∼300 ns (Figure 6A). The latter corresponds to a rotation of one monomer with respect to the other (Video S6), similar to the E. coli DHDPS dimer (Figure 2B). Active site residues deviate moderately from their crystal conformation over the course of the simulations (Figure 6B and Video S6), reaching a plateau for the last 200 ns, yet somewhat less deviant than the corresponding residues in the E. coli DHDPS dimer (RMSD values of 1.6–3.0 Å compared to 2.2–3.5 Å; Figure 6B).


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)

A detailed view of the tight-dimer interface in E. coli and MRSA DHDPS.Surfaces of both enzymes with the residues involved in the tight-dimer interface represented in light orange. Residues involved in hydrogen bonds are shown in red and in salt-bridges in yellow, as calculated by the PISA server (A) Dimer from E. coli wild-type tetramer (PDB ID: 1YXC); (B) MRSA wild-type dimer (PDB ID: 3DAQ).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002537-g005: A detailed view of the tight-dimer interface in E. coli and MRSA DHDPS.Surfaces of both enzymes with the residues involved in the tight-dimer interface represented in light orange. Residues involved in hydrogen bonds are shown in red and in salt-bridges in yellow, as calculated by the PISA server (A) Dimer from E. coli wild-type tetramer (PDB ID: 1YXC); (B) MRSA wild-type dimer (PDB ID: 3DAQ).
Mentions: Our simulation data for E. coli DHDPS suggest that conformational fluctuations and flexibility at the active site is a primary cause of the dramatic decrease in enzymatic activity of dimers. The existence of a naturally occurring dimer from the bacterial pathogen MRSA that exhibits comparable activity to the E. coli tetramer is therefore intriguing [8]. Whereas the overall tertiary structures of MRSA and E. coli DHDPS are highly similar (RMSD = 0.9 Å; Figure 1B,C), with only minor reorientations of active site side-chains (Figure 1D), the nature of their tight-dimer interfaces differs (Figure 5). MRSA DHDPS possesses a relatively high number of hydrogen bonds at the tight-dimer interface and two electrostatic interactions that are absent in the E. coli structure, suggesting that it is perhaps less flexible that its E. coli counterpart [8]. We therefore performed two MD simulations of the MRSA DHDPS dimer in the absence of substrate and compared the results to the E. coli DHDPS simulations. The 1.45 Å resolution crystal structure of MRSA DHDPS [8] was used as the starting structure for two independent MD simulations of 0.5 µs each in length (denoted mrsa-1 and mrsa-2). Both simulations show a gradual increase in RMSD, which stabilise and reach a plateau at ∼3 Å at ∼300 ns (Figure 6A). The latter corresponds to a rotation of one monomer with respect to the other (Video S6), similar to the E. coli DHDPS dimer (Figure 2B). Active site residues deviate moderately from their crystal conformation over the course of the simulations (Figure 6B and Video S6), reaching a plateau for the last 200 ns, yet somewhat less deviant than the corresponding residues in the E. coli DHDPS dimer (RMSD values of 1.6–3.0 Å compared to 2.2–3.5 Å; Figure 6B).

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