Dataset Information

Engineering Specificity from Broad to Narrow: Design of a ?-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC ?-Lactamase.

ABSTRACT: The ?-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A ?-lactamases including the TEM-1 ?-lactamase (Ki = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 ?-lactamase (Ki = 1.2 nM), which hydrolyzes virtually all clinically useful ?-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 ?-lactamase. A genetic screen for BLIP function in Escherichia coli was used to narrow the binding specificity of BLIP by identifying amino acid substitutions that retain affinity for KPC-2 while losing affinity for TEM-1 ?-lactamase. The combination of single substitutions yielded the K74T:W112D BLIP variant, which was shown by inhibition assays to retain high affinity for KPC-2 with a Ki of 0.4 nM, while drastically losing affinity for TEM-1 with a Ki > 10 ?M. The K74T:W112D mutant therefore binds KPC-2 ?-lactamase 3 times more tightly while binding TEM-1 > 20000-fold more weakly than wild-type BLIP. The K74T:W112D BLIP variant also exhibited low affinity (Ki > 10 ?M) for other class A ?-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 ?-lactamase suggest it could be a useful sensor for the presence of this enzyme in multidrug-resistant bacteria. This was demonstrated with an assay employing BLIP K74T:W112D conjugated to a bead to specifically pull-down and detect KPC-2 ?-lactamase in lysates from clinical bacterial isolates containing multiple ?-lactamases.

SUBMITTER: Chow DC

PROVIDER: S-EPMC5656002 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase.

Chow Dar-Chone DC Rice Kacie K Huang Wanzhi W Atmar Robert L RL Palzkill Timothy T

ACS infectious diseases 20161026 12

The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (K<sub>i</sub> = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (K<sub>i</sub> = 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineeri ...[more]

PMID: 27756125

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Project description:KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Dataset Information

Engineering Specificity from Broad to Narrow: Design of a ?-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC ?-Lactamase.

Publications

Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase.

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