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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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Second-order rates ofacylation of Bocillin-FL against full-lengthand truncated constructs of wild-type PBP2 and PBP2 from the penicillin-resistantstrain 6140. For each experiment, the pseudo-first-order rates ofacylation (ka) were plotted vs the concentrationof Bocillin-FL. Shown is the average plot of at least two or moreindependent experiments, where the slope of the line yields the second-order k2/Ks value.
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fig1: Second-order rates ofacylation of Bocillin-FL against full-lengthand truncated constructs of wild-type PBP2 and PBP2 from the penicillin-resistantstrain 6140. For each experiment, the pseudo-first-order rates ofacylation (ka) were plotted vs the concentrationof Bocillin-FL. Shown is the average plot of at least two or moreindependent experiments, where the slope of the line yields the second-order k2/Ks value.

Mentions: Before we proceeded with crystallization trials, it was importantto determine whether the absence of the N-terminal domain impactedthe acylation rate of the truncated PBP2 constructs. The second-orderrates of acylation (k2/Ks) of (full-length) wild-type PBP2 and PBP2-t3-wt forthe fluorescent penicillin, Bocillin-FL, were nearly identical, andthe corresponding k2/Ks acylation rates for PBP2-6140 (full-length PBP2 containingfive mutations) and PBP2-t3-6140 are also very similar (Table 1 and Figure 1). For bothfull-length and truncated constructs, the fold difference betweenthe wild type (14-fold) and 6140 (9-fold) was also similar. Thesedata indicate that removal of the N-terminal domain does not significantlyimpact the β-lactam-binding activity of the TPase domain andthat mutations associated with penicillin resistance have the sameimpact on the acylation kinetics.


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)

Second-order rates ofacylation of Bocillin-FL against full-lengthand truncated constructs of wild-type PBP2 and PBP2 from the penicillin-resistantstrain 6140. For each experiment, the pseudo-first-order rates ofacylation (ka) were plotted vs the concentrationof Bocillin-FL. Shown is the average plot of at least two or moreindependent experiments, where the slope of the line yields the second-order k2/Ks value.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Second-order rates ofacylation of Bocillin-FL against full-lengthand truncated constructs of wild-type PBP2 and PBP2 from the penicillin-resistantstrain 6140. For each experiment, the pseudo-first-order rates ofacylation (ka) were plotted vs the concentrationof Bocillin-FL. Shown is the average plot of at least two or moreindependent experiments, where the slope of the line yields the second-order k2/Ks value.
Mentions: Before we proceeded with crystallization trials, it was importantto determine whether the absence of the N-terminal domain impactedthe acylation rate of the truncated PBP2 constructs. The second-orderrates of acylation (k2/Ks) of (full-length) wild-type PBP2 and PBP2-t3-wt forthe fluorescent penicillin, Bocillin-FL, were nearly identical, andthe corresponding k2/Ks acylation rates for PBP2-6140 (full-length PBP2 containingfive mutations) and PBP2-t3-6140 are also very similar (Table 1 and Figure 1). For bothfull-length and truncated constructs, the fold difference betweenthe wild type (14-fold) and 6140 (9-fold) was also similar. Thesedata indicate that removal of the N-terminal domain does not significantlyimpact the β-lactam-binding activity of the TPase domain andthat mutations associated with penicillin resistance have the sameimpact on the acylation kinetics.

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