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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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Related in: MedlinePlus

Superimposition of the structures of PBP2-t3-6140 and the TPase/β-lactam-bindingdomain from wild-type PBP2. In this stereoview, each molecule is displayedin cartoon form, with PBP2-t3-6140 colored blue and wild-type PBP2colored yellow. Regions exhibiting structural differences are indicated.The β-hairpin region of PBP2-t3-6140 is colored green. The locationof the Asp insertion is indicated by a red sphere, corresponding tothe Cα position, and that of Ser310 by a purple sphere.
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fig4: Superimposition of the structures of PBP2-t3-6140 and the TPase/β-lactam-bindingdomain from wild-type PBP2. In this stereoview, each molecule is displayedin cartoon form, with PBP2-t3-6140 colored blue and wild-type PBP2colored yellow. Regions exhibiting structural differences are indicated.The β-hairpin region of PBP2-t3-6140 is colored green. The locationof the Asp insertion is indicated by a red sphere, corresponding tothe Cα position, and that of Ser310 by a purple sphere.

Mentions: Aside from the insertionsite and the aforementioned β3−β4 loop, the otherdifferences are minimal (Figure 4). The shiftin residues 388–403, which is a β-hairpin loop that connectsα9 with α10, can be explained by the absence of the N-terminaldomain because, in the structure of full-length PBP2, this loop packsdirectly against the N-terminal domain and those contacts are lostin PBP2-t3-6140. In fact, this is the only difference between thetwo structures that can be attributed directly to the absence of theN-terminal domain. As expected, there is also a difference at thePro282-Arg298 “join” in PBP2-t3-6140. The removal ofresidues 283–297 (with addition of a Gly linker), however,was a very successful strategy because there is only a marginal differencein the structure immediately preceding and following the join. Thejoin itself exhibits excellent density, even though the Gly linkeris non-native (Figure 3 of the Supporting Information).


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)

Superimposition of the structures of PBP2-t3-6140 and the TPase/β-lactam-bindingdomain from wild-type PBP2. In this stereoview, each molecule is displayedin cartoon form, with PBP2-t3-6140 colored blue and wild-type PBP2colored yellow. Regions exhibiting structural differences are indicated.The β-hairpin region of PBP2-t3-6140 is colored green. The locationof the Asp insertion is indicated by a red sphere, corresponding tothe Cα position, and that of Ser310 by a purple sphere.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Superimposition of the structures of PBP2-t3-6140 and the TPase/β-lactam-bindingdomain from wild-type PBP2. In this stereoview, each molecule is displayedin cartoon form, with PBP2-t3-6140 colored blue and wild-type PBP2colored yellow. Regions exhibiting structural differences are indicated.The β-hairpin region of PBP2-t3-6140 is colored green. The locationof the Asp insertion is indicated by a red sphere, corresponding tothe Cα position, and that of Ser310 by a purple sphere.
Mentions: Aside from the insertionsite and the aforementioned β3−β4 loop, the otherdifferences are minimal (Figure 4). The shiftin residues 388–403, which is a β-hairpin loop that connectsα9 with α10, can be explained by the absence of the N-terminaldomain because, in the structure of full-length PBP2, this loop packsdirectly against the N-terminal domain and those contacts are lostin PBP2-t3-6140. In fact, this is the only difference between thetwo structures that can be attributed directly to the absence of theN-terminal domain. As expected, there is also a difference at thePro282-Arg298 “join” in PBP2-t3-6140. The removal ofresidues 283–297 (with addition of a Gly linker), however,was a very successful strategy because there is only a marginal differencein the structure immediately preceding and following the join. Thejoin itself exhibits excellent density, even though the Gly linkeris non-native (Figure 3 of the Supporting Information).

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