期刊
ACS INFECTIOUS DISEASES
卷 2, 期 12, 页码 81-91出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsinfecdis.6b00160
关键词
protein-protein interactions; protein engineering antibiotic resistance; beta-lactamase; enzyme inhibition; diagnostics
资金
- National Institutes of Health [AI092963, AI32956]
The beta-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A beta-lactamases including the TEM-1 beta-lactamase (K-i = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 beta-lactamase (K-i = 1.2 nM), which hydrolyzes virtually all clinically useful beta-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 beta-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 beta-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 K-i > 10 mu M. The K74T:W112D mutant therefore binds KPC-2 beta-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 (K, > 10 itM) for other class A fl-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 beta-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 beta-lactamase in lysates from clinical bacterial isolates containing multiple beta-lactamases.
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