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Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

Fedarovich A, Cook E, Tomberg J, Nicholas RA, Davies C - Biochemistry (2014)

Bottom Line: Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function.The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif.The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina , Charleston, South Carolina 29425, United States.

ABSTRACT
A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c-β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

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Stereoviewof a superimposition of the structures of PBP2-t3-6140and wild-type PBP2 in which a meropenem molecule was docked via superimpositionwith a crystal structure of PBP2 in complex with meropenem. Wild-typePBP2 is colored yellow, PBP2-t3-6140 blue, and meropenem orange. Thehydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red. The three conserved active-sitemotifs of PBPs are labeled in blue.
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fig7: Stereoviewof a superimposition of the structures of PBP2-t3-6140and wild-type PBP2 in which a meropenem molecule was docked via superimpositionwith a crystal structure of PBP2 in complex with meropenem. Wild-typePBP2 is colored yellow, PBP2-t3-6140 blue, and meropenem orange. Thehydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red. The three conserved active-sitemotifs of PBPs are labeled in blue.

Mentions: By lengtheningthe β2c−β2d loop, the primary effect of the insertionis to project the new aspartate side chain directly toward the activesite, potentially placing it within hydrogen bonding distance of Asn364.The latter is part of the SxN active-site motif that is found in allserine-based PBPs and β-lactamases. The mechanistic role ofAsn364 in both transpeptidation and β-lactam binding is notentirely clear, but it is required for activity27 and participates in the rich hydrogen bonding environmentwithin the active site. This network involves both Ser310, which isthe nucleophile that forms the acyl–enzyme bond with peptidesubstrates and β-lactam antibiotics, and Lys313, which is believedto activate the serine nucleophile by functioning in PBPs as a generalbase.26,28−30 Although disruptionof the hydrogen bonding network by the inserted Asp might be responsiblefor the decreased rates of acylation with β-lactams, the closeoverlap of active-site residues in both the wild-type and 6140 structuresof PBP2 shows the hydrogen bonding is essentially the same in thepresence of the insertion (Figure 7). The apparentflexibility around the Asp insertion site and with it the absenceof a stable hydrogen bond with Asn364 also make this mechanism uncertain.


Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

Fedarovich A, Cook E, Tomberg J, Nicholas RA, Davies C - Biochemistry (2014)

Stereoviewof a superimposition of the structures of PBP2-t3-6140and wild-type PBP2 in which a meropenem molecule was docked via superimpositionwith a crystal structure of PBP2 in complex with meropenem. Wild-typePBP2 is colored yellow, PBP2-t3-6140 blue, and meropenem orange. Thehydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red. The three conserved active-sitemotifs of PBPs are labeled in blue.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263433&req=5

fig7: Stereoviewof a superimposition of the structures of PBP2-t3-6140and wild-type PBP2 in which a meropenem molecule was docked via superimpositionwith a crystal structure of PBP2 in complex with meropenem. Wild-typePBP2 is colored yellow, PBP2-t3-6140 blue, and meropenem orange. Thehydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red. The three conserved active-sitemotifs of PBPs are labeled in blue.
Mentions: By lengtheningthe β2c−β2d loop, the primary effect of the insertionis to project the new aspartate side chain directly toward the activesite, potentially placing it within hydrogen bonding distance of Asn364.The latter is part of the SxN active-site motif that is found in allserine-based PBPs and β-lactamases. The mechanistic role ofAsn364 in both transpeptidation and β-lactam binding is notentirely clear, but it is required for activity27 and participates in the rich hydrogen bonding environmentwithin the active site. This network involves both Ser310, which isthe nucleophile that forms the acyl–enzyme bond with peptidesubstrates and β-lactam antibiotics, and Lys313, which is believedto activate the serine nucleophile by functioning in PBPs as a generalbase.26,28−30 Although disruptionof the hydrogen bonding network by the inserted Asp might be responsiblefor the decreased rates of acylation with β-lactams, the closeoverlap of active-site residues in both the wild-type and 6140 structuresof PBP2 shows the hydrogen bonding is essentially the same in thepresence of the insertion (Figure 7). The apparentflexibility around the Asp insertion site and with it the absenceof a stable hydrogen bond with Asn364 also make this mechanism uncertain.

Bottom Line: Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function.The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif.The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina , Charleston, South Carolina 29425, United States.

ABSTRACT
A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c-β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

Show MeSH
Related in: MedlinePlus