An insight into the biophysical characterization of different states of cefotaxime hydrolyzing β-lactamase 15 (CTX-M-15)

Abstract

Cefotaxime hydrolyzing β-lactamase-15 (CTX-M-15) is encoded by bla_CTX-M-15 gene present on plasmid of various Gram-negative bacteria, such as E. coli, E. cloacae, K. pneumoniae, etc. The widespread dissemination of CTX-M-15 harboring bacteria in hospital as well as community settings is a universal threat as they are resistant to various clinically significant antibiotics. In order to gain an insight into the folding mechanism of CTX-M-15, we carried out pH-induced denaturation study by monitoring Trp fluorescence, far-UV circular dichroism (CD), and ANS fluorescence. We found that the pH-induced denaturation of CTX-M-15 was a three-step process with the accumulation of two stable folding intermediates (X_I at pH 2.5 and X_II at pH 1.5) in the folding pathway. The intermediates were further characterized by far-UV and near-UV CD analysis, Trp fluorescence, ANS fluorescence, three-dimensional fluorescence, acrylamide quenching, dynamic light scattering, and thermal denaturation studies. We found that X_I state lacked tertiary structure but retained most of the secondary structure, its Trp residues were partially exposed to the solvent and its hydrophobic patches were highly accessible to ANS. On the other hand, a complete disruption of tertiary structure along with more than 50% loss in secondary structure was observed in X_II state. We conclude that the X_I state of CTX-M-15 at pH 2.5 had all the characteristics of a molten globule (MG) state, while its X_II state at pH 1.5 was more similar to pre-molten globule (PMG) state. ANS fluorescence also showed that the binding of ANS in X_II state was lower than that in the X_I state. We propose that the accumulation of MG- and PMG-states was due to separation (at pH 2.5) and then unfolding (at pH 1.5) of the αβα-fold of CTX-M-15, respectively.

Keywords:

• protein folding
• protein stability
• three-dimensional fluorescence
• non-native intermediates
• extended spectrum β-lactamase

Acknowledgments

UGC is sincerely accredited for financial assistance in the form of Dr D S Kothari Postdoctoral Fellowship to MTR. MF is thankful to ICMR for SRF.