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

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Protein stability is crucial for biological function, but in-cell stability factors remain poorly understood.
• Metallo-β-lactamases (MBLs) are important in antibiotic resistance.
• New Delhi metallo-β-lactamase 1 (NDM-1) is a significant MBL.

Purpose of the Study:

• To investigate the factors governing the in-cell stability of NDM-1.
• To understand how NDM-1 optimizes its stability within the cell.
• To explore the link between NDM-1 stability and antibiotic resistance.

Main Methods:

• In vitro and in-cell stability assays.
• Analysis of NDM-1 degradation by specific proteases (Prc and DegP).
• Investigating the role of metal binding (Zn(II)) and membrane anchoring on NDM-1 stability.
• Studying NDM variants with C-terminal substitutions.

Main Results:

• Apo (non-metalated) NDM-1 is kinetically unstable and degraded by the periplasmic protease Prc due to its flexible C-terminal domain.
• Zinc(II) binding stabilizes NDM-1 by reducing C-terminal flexibility, preventing degradation.
• Membrane anchoring protects apo-NDM-1 from Prc and DegP.
• NDM variants with C-terminal substitutions exhibit enhanced kinetic stability and resistance to proteolysis.

Conclusions:

• NDM-1 has evolved biochemical traits, including metal binding and C-terminal modifications, to enhance its in-cell stability.
• These stability mechanisms are crucial for NDM-1 function and likely contribute to MBL-mediated antibiotic resistance.
• The findings highlight the importance of cellular protein homeostasis in regulating MBL activity and resistance.