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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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Stereoviewshowing the superimposition of wild-type PBP2 (yellow)and PBP2-t3-6140 (blue). Molecule A of each structure was superimposed.The hydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red.
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fig6: Stereoviewshowing the superimposition of wild-type PBP2 (yellow)and PBP2-t3-6140 (blue). Molecule A of each structure was superimposed.The hydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red.

Mentions: A second observationis that the insertion is best described functionallyas an Asp346a insertion. As shown by superimposition with the structureof wild-type PBP2 (Figure 6), the structuralshift occurs after Asp346, which is essentially unchanged in positionand makes the same hydrogen bonding interaction with Ser363 of theSxN motif that is present in wild-type PBP2. Finally, the β2a−β2dhairpin loop has shifted relatively little, with the biggest differencebeing a longer loop between β2b and β2c as a result ofthe additional residue. Overall, these structural observations agreeclosely with our previous mutagenesis data that show the effect ofthe Asp insertion to be highly specific.18


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)

Stereoviewshowing the superimposition of wild-type PBP2 (yellow)and PBP2-t3-6140 (blue). Molecule A of each structure was superimposed.The hydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Stereoviewshowing the superimposition of wild-type PBP2 (yellow)and PBP2-t3-6140 (blue). Molecule A of each structure was superimposed.The hydrogen bond between Asp346 and Ser363 is shown as a dashed line,and the inserted Asp is labeled in red.
Mentions: A second observationis that the insertion is best described functionallyas an Asp346a insertion. As shown by superimposition with the structureof wild-type PBP2 (Figure 6), the structuralshift occurs after Asp346, which is essentially unchanged in positionand makes the same hydrogen bonding interaction with Ser363 of theSxN motif that is present in wild-type PBP2. Finally, the β2a−β2dhairpin loop has shifted relatively little, with the biggest differencebeing a longer loop between β2b and β2c as a result ofthe additional residue. Overall, these structural observations agreeclosely with our previous mutagenesis data that show the effect ofthe Asp insertion to be highly specific.18

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