In-cell kinetic stability is an essential trait in metallo-β-lactamase evolution

Protein stability is an essential property for biological function. In contrast to the vast knowledge on protein stability in vitro, little is known about the factors governing in-cell stability. Here we show that the metallo-beta-lactamase (MBL) New Delhi MBL-1 (NDM-1) is a kinetically unstable protein on metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability. The nonmetalated (apo) NDM-1 is degraded by the periplasmic protease Prc that recognizes its partially unstructured C-terminal domain. 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. NDM variants accumulate substitutions at the C terminus that quench its flexibility, enhancing their kinetic stability and bypassing proteolysis. These observations link MBL-mediated resistance with the essential periplasmic metabolism, highlighting the importance of the cellular protein homeostasis.

Automated summary

Protein stability is crucial for biological function. This study reveals that the metallo-beta-lactamase New Delhi MBL-1 (NDM-1) is unstable in the absence of metal, but has evolved biochemical traits to optimize its stability inside cells. Zn(II) binding and membrane anchoring protect NDM-1 from degradation by cellular proteases. NDM variants accumulate substitutions at the C terminus that enhance their stability. This research highlights the importance of cellular protein homeostasis and its connection with antibiotic resistance.