Metallo-β-lactamases (MBLs) are zinc-dependent hydrolases able to degrade almost all β-lactam antibiotics. Among them, the New Delhi MBL-1 (NDM-1) has experienced a worldwide dissemination. Their resistance phenotype depends on the zinc cargo in the periplasm of Gram negative bacteria. The host native immune system response limits the availability of the Zn(II) ions at the infection sites, leading to accumulation of the non-metalated (apo) NDM-1 variant in the periplasm, that is degraded by the periplasmic protease Prc by recognition of a partially unstructured C-terminal domain. Accumulation of misfolded apoNDM-1 is further targeted by the canonical housekeeping protease DegP.
Therefore, NDM-1 is a kinetically unstable protein upon metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability. Zn(II) binding renders the protein refractory to degradation by quenching the flexibility of this region. Membrane anchoring makes apo-NDM-1 less accessible to Prc and protects it from DegP, a cellular protease degrading misfolded, nonmetalated NDM-1 precursors. Clinical NDM variants accumulate substitutions at the C terminus that quench its flexibility, enhancing their kinetic stability and bypassing proteolysis.
We studied the degradation of the New Delhi metallo-β-lactamase (NDM-1) in the bacterial periplasm of E. coli by using in-cell NMR to simultaneously delineate the degradation of NDM-1 by Prc and DegP. We identified the cleavage sites of each protease and their concerted mechanism of action providing new insights about the molecular recognition events in living E. coli cells.
Our initiative highlights the potential of in-cell NMR to characterize molecular networks within the cell, in a highly challenging subcellular compartment such as the bacterial periplasm