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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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Electron density of the β3−β4 loop in each moleculeof the asymmetric unit. The 2/Fo/ –/Fc/ electron density is contoured at1σ. Sites of mutation compared with PBP2 from penicillin-susceptiblestrains of N. gonorrhoeae are labeled in red. Arrowsindicate the preceding and following polypeptide chains: (A) moleculeA and (B) molecule B.
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fig3: Electron density of the β3−β4 loop in each moleculeof the asymmetric unit. The 2/Fo/ –/Fc/ electron density is contoured at1σ. Sites of mutation compared with PBP2 from penicillin-susceptiblestrains of N. gonorrhoeae are labeled in red. Arrowsindicate the preceding and following polypeptide chains: (A) moleculeA and (B) molecule B.

Mentions: Overall, the two molecules in the asymmetric unit adopt similarstructures and superimpose with a rmsd of 0.86 Å for all commonmain chain atoms (Figure 2). There are, however,some structural differences of note. In the crystal structures ofboth wild-type PBP2 and PBP2-6140CT (which contains the four C-terminalsubstitutions but lacks the Asp345a insertion), 10–11 residuesof the β3−β4 loop could not be modeled becauseof flexibility.17 In molecule A of thecrystal structure of PBP2-t3-6140, all residues of the loop are visiblein the electron density, thus providing the first structural viewof these residues (Figure 3). In contrast,residues 504–511 in molecule B exhibit weak or absent electrondensity and could not be modeled. This region is significant becausetwo mutations associated with penicillin resistance (P504L and A510V)reside in this loop.17 The differencesin this region between the two molecules of the asymmetric unit suggestthe degree of flexibility in the β3−β4 loop isinfluenced directly by crystal packing interactions. Hence, the structuralimpact on this loop of mutations associated with antibiotic resistancemust be assessed with caution. Other regions that differ in the twomolecules of the asymmetric unit are the β2a−β2dhairpin, the loop between α9 and α10, the N-terminal endof α8, and the β5−α11 loop (Figure 2). As features on the surface of the molecule, allthese differences could result from the different crystal packingenvironment around each molecule.


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)

Electron density of the β3−β4 loop in each moleculeof the asymmetric unit. The 2/Fo/ –/Fc/ electron density is contoured at1σ. Sites of mutation compared with PBP2 from penicillin-susceptiblestrains of N. gonorrhoeae are labeled in red. Arrowsindicate the preceding and following polypeptide chains: (A) moleculeA and (B) molecule B.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Electron density of the β3−β4 loop in each moleculeof the asymmetric unit. The 2/Fo/ –/Fc/ electron density is contoured at1σ. Sites of mutation compared with PBP2 from penicillin-susceptiblestrains of N. gonorrhoeae are labeled in red. Arrowsindicate the preceding and following polypeptide chains: (A) moleculeA and (B) molecule B.
Mentions: Overall, the two molecules in the asymmetric unit adopt similarstructures and superimpose with a rmsd of 0.86 Å for all commonmain chain atoms (Figure 2). There are, however,some structural differences of note. In the crystal structures ofboth wild-type PBP2 and PBP2-6140CT (which contains the four C-terminalsubstitutions but lacks the Asp345a insertion), 10–11 residuesof the β3−β4 loop could not be modeled becauseof flexibility.17 In molecule A of thecrystal structure of PBP2-t3-6140, all residues of the loop are visiblein the electron density, thus providing the first structural viewof these residues (Figure 3). In contrast,residues 504–511 in molecule B exhibit weak or absent electrondensity and could not be modeled. This region is significant becausetwo mutations associated with penicillin resistance (P504L and A510V)reside in this loop.17 The differencesin this region between the two molecules of the asymmetric unit suggestthe degree of flexibility in the β3−β4 loop isinfluenced directly by crystal packing interactions. Hence, the structuralimpact on this loop of mutations associated with antibiotic resistancemust be assessed with caution. Other regions that differ in the twomolecules of the asymmetric unit are the β2a−β2dhairpin, the loop between α9 and α10, the N-terminal endof α8, and the β5−α11 loop (Figure 2). As features on the surface of the molecule, allthese differences could result from the different crystal packingenvironment around each molecule.

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