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

Changes at the tight-dimer interface during simulations.(A). Interfacial surface area buried for both monomers; (B) Number of interfacial hydrogen-bonds (tet-1:black; dim-A: blue; mrsa-1: green). Spline fits (thick lines) of the values (thin lines) are represented for clarity.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002537-g008: Changes at the tight-dimer interface during simulations.(A). Interfacial surface area buried for both monomers; (B) Number of interfacial hydrogen-bonds (tet-1:black; dim-A: blue; mrsa-1: green). Spline fits (thick lines) of the values (thin lines) are represented for clarity.

Mentions: Whereas the E. coli DHDPS dimer interface consists of seven hydrogen bonds and three hydrophobic contacts, the larger MRSA DHDPS dimer interface consists of 17 hydrogen bonds and two salt-bridges [8]. We therefore compared and contrasted the nature of the tight-dimer interfaces for E. coli. and MRSA enzymes. The size of the interfacial area in the E. coli tetramer is stable throughout the simulations. We find that in the MRSA dimer the rotation of the monomers is associated with a reduction in the buried interfacial area, similar in size (∼2700 Å2 for two monomers, Figure 8A) to the initial E. coli interface. This does not lead to a decrease in the number of hydrogen bonds (Figure 8B) or salt-bridges, which remains constant. We find however that in the mutant E. coli dimer, while the interfacial buried area is constant, the number of hydrogen bonds contributing to the tight-dimer interface increases with re-orientation of the monomers. In addition we observed the formation of a new salt-bridge per monomer between residues R109 and E246 in dim-A and dim-B, permitted by the new orientation of the monomers. In mrsa-1 and mrsa-2 the equivalent salt-bridge is formed at positions K111 and D247. This suggests that this re-organization of the monomers is more stable than the arrangement found in the crystal state but only compatible with loss of the quaternary structure. Dimer binding energies calculated by the MM-PBSA approach lend support to this hypothesis (Text S1). Disruption of the supra-molecular assembly is associated in E. coli DHDPS with dramatic conformational changes in the active site.


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)

Changes at the tight-dimer interface during simulations.(A). Interfacial surface area buried for both monomers; (B) Number of interfacial hydrogen-bonds (tet-1:black; dim-A: blue; mrsa-1: green). Spline fits (thick lines) of the values (thin lines) are represented for clarity.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002537-g008: Changes at the tight-dimer interface during simulations.(A). Interfacial surface area buried for both monomers; (B) Number of interfacial hydrogen-bonds (tet-1:black; dim-A: blue; mrsa-1: green). Spline fits (thick lines) of the values (thin lines) are represented for clarity.
Mentions: Whereas the E. coli DHDPS dimer interface consists of seven hydrogen bonds and three hydrophobic contacts, the larger MRSA DHDPS dimer interface consists of 17 hydrogen bonds and two salt-bridges [8]. We therefore compared and contrasted the nature of the tight-dimer interfaces for E. coli. and MRSA enzymes. The size of the interfacial area in the E. coli tetramer is stable throughout the simulations. We find that in the MRSA dimer the rotation of the monomers is associated with a reduction in the buried interfacial area, similar in size (∼2700 Å2 for two monomers, Figure 8A) to the initial E. coli interface. This does not lead to a decrease in the number of hydrogen bonds (Figure 8B) or salt-bridges, which remains constant. We find however that in the mutant E. coli dimer, while the interfacial buried area is constant, the number of hydrogen bonds contributing to the tight-dimer interface increases with re-orientation of the monomers. In addition we observed the formation of a new salt-bridge per monomer between residues R109 and E246 in dim-A and dim-B, permitted by the new orientation of the monomers. In mrsa-1 and mrsa-2 the equivalent salt-bridge is formed at positions K111 and D247. This suggests that this re-organization of the monomers is more stable than the arrangement found in the crystal state but only compatible with loss of the quaternary structure. Dimer binding energies calculated by the MM-PBSA approach lend support to this hypothesis (Text S1). Disruption of the supra-molecular assembly is associated in E. coli DHDPS with dramatic conformational changes in the active site.

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