Distant site mutations in clinical TEM β-lactamase variants enhance non-covalent binding to ceftazidime: Insights from biophysical and in silico investigations

Abstract PurposeThis study investigated the bioactive interactions of ceftazidime (CAZ) to TEM β-lactamase variants with distant site mutations isolated from clinical settings to explore the cause of their selection and dissemination due to empirical use of β-lactams.MethodsBinding interactions of CAZ with wild-type and mutant (M184, M203, M210) TEM β-lactamases were recorded by UV–Vis absorption and fluorescence spectra (280–600 nm) at 298–311 K with inner filter correction. Binding constants were determined using Stern–Volmer and modified Stern–Volmer equations. Fluorescence lifetime, quantum yield and anisotropy measurements ascertained energy transfer and microenvironmental changes. Structural alterations and interaction energetics were evaluated using circular dichroism, Raman spectroscopy, Förster resonance energy transfer and Molecular dynamics simulation (100 ns) study.ResultsBiophysical experimentation indicated facilitated binding of CAZ to the active site of the mutants than the wild type. The CAZ-TEM β-lactamase mutant interactions were predominantly hydrophobic compared to H-bonding and van der Waals forces in the CAZ-wild type complex. Additionally, structural alteration to justify more rigid binding of CAZ to the mutants in contrast to the wild type enzyme was established in silico. Acquisition of distant site mutations with respect to the active site of TEM β-lactamases rendered conformational flexibility to accommodate CAZ was evidenced.ConclusionTherefore, this study established that selection of far site mutations in TEM β-lactamases can indirectly contribute to β-lactam resistance by optimizing substrate binding dynamics in response to rampant usage of the antibiotics and provided a new dimension towards future drug development.