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Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics.

Boudet J, Duval V, Van Melckebeke H, Blackledge M, Amoroso A, Joris B, Simorre JP - Nucleic Acids Res. (2007)

Bottom Line: To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner.Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA.These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean-Pierre Ebel CEA-CNRS-UJF, 41 Avenue Jules Horowitz, 38027 Grenoble Cedex 1, France.

ABSTRACT
In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

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Ten lowest energy structures obtained by de novo docking of the Bacillus licheniformis BlaI-NTD in interaction with the blaP semi-operating sequence. (A) Front view and (B) back view of the structure ribbon representation. Root mean square deviation has been calculated to 0.7Å for all heavy atoms of the 10 structures. The 12 bp DNA of blaP half operator are shown in orange. H3 helix is inserted into the major groove and the minor groove is overhung by the wing motif.
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Figure 3: Ten lowest energy structures obtained by de novo docking of the Bacillus licheniformis BlaI-NTD in interaction with the blaP semi-operating sequence. (A) Front view and (B) back view of the structure ribbon representation. Root mean square deviation has been calculated to 0.7Å for all heavy atoms of the 10 structures. The 12 bp DNA of blaP half operator are shown in orange. H3 helix is inserted into the major groove and the minor groove is overhung by the wing motif.

Mentions: Ten lowest energy structures from 250 calculations of the [15N-13C BLBlaI-NTD]/[1/2OP1blaP] have been generated using a de novo driven docking (Figure 3). As expected, the global fold of the NTD repressor domain is not modified by the complex formation. Pairwise RMSD calculated on the NTD backbone atoms between the monomeric complexed BLBlaI structure and free BLBlaI-NTD, SAMecI/OPblaZ and SABlaI/OPblaZ is 1, 1.6 and 1.5 Å, respectively. The typical structural arrangements of the WHP family protein is conserved without violations in the structure calculation file. The three α-helices H1 (9–20), H2 (26–36), H3 (41–54) and the three stranded β-sheets S1 (23–25), S2 (57–62), S3 (65–70) are packed following the sequence H1–S1–H2–H3–W1–S2–S3. The wing motif W1 consists of a short loop (residues 63 and 64). Three-dimensional structures of the free and bound forms of the BLBlaI-NTD are very close except for the more dynamic residues located in the N- and C-terminals extremities of the protein, namely M1 to I4 and Y77 to S82, respectively. Moreover, it should be noticed that restricted conformational modification occurred for the residues of the Wing (G63 and R64) and for few residues surrounding this motif (E62, V65 and F66).Figure 3.


Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics.

Boudet J, Duval V, Van Melckebeke H, Blackledge M, Amoroso A, Joris B, Simorre JP - Nucleic Acids Res. (2007)

Ten lowest energy structures obtained by de novo docking of the Bacillus licheniformis BlaI-NTD in interaction with the blaP semi-operating sequence. (A) Front view and (B) back view of the structure ribbon representation. Root mean square deviation has been calculated to 0.7Å for all heavy atoms of the 10 structures. The 12 bp DNA of blaP half operator are shown in orange. H3 helix is inserted into the major groove and the minor groove is overhung by the wing motif.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935004&req=5

Figure 3: Ten lowest energy structures obtained by de novo docking of the Bacillus licheniformis BlaI-NTD in interaction with the blaP semi-operating sequence. (A) Front view and (B) back view of the structure ribbon representation. Root mean square deviation has been calculated to 0.7Å for all heavy atoms of the 10 structures. The 12 bp DNA of blaP half operator are shown in orange. H3 helix is inserted into the major groove and the minor groove is overhung by the wing motif.
Mentions: Ten lowest energy structures from 250 calculations of the [15N-13C BLBlaI-NTD]/[1/2OP1blaP] have been generated using a de novo driven docking (Figure 3). As expected, the global fold of the NTD repressor domain is not modified by the complex formation. Pairwise RMSD calculated on the NTD backbone atoms between the monomeric complexed BLBlaI structure and free BLBlaI-NTD, SAMecI/OPblaZ and SABlaI/OPblaZ is 1, 1.6 and 1.5 Å, respectively. The typical structural arrangements of the WHP family protein is conserved without violations in the structure calculation file. The three α-helices H1 (9–20), H2 (26–36), H3 (41–54) and the three stranded β-sheets S1 (23–25), S2 (57–62), S3 (65–70) are packed following the sequence H1–S1–H2–H3–W1–S2–S3. The wing motif W1 consists of a short loop (residues 63 and 64). Three-dimensional structures of the free and bound forms of the BLBlaI-NTD are very close except for the more dynamic residues located in the N- and C-terminals extremities of the protein, namely M1 to I4 and Y77 to S82, respectively. Moreover, it should be noticed that restricted conformational modification occurred for the residues of the Wing (G63 and R64) and for few residues surrounding this motif (E62, V65 and F66).Figure 3.

Bottom Line: To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner.Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA.These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean-Pierre Ebel CEA-CNRS-UJF, 41 Avenue Jules Horowitz, 38027 Grenoble Cedex 1, France.

ABSTRACT
In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

Show MeSH
Related in: MedlinePlus