-IN-CELL KINETIC STABILITY IS AN ESSENTIAL TRAIT IN THE EVOLUTION OF METALLO-ΒLACTAMASES IN BACTERIA

GONZALEZ LJ; BAHR G; GONZÁLEZ MM; VILA AJ

Los Cocos

Congreso; XVII Congreso Argentino de Microbiología General; 2022

SAMIGE

Protein stability is an essential property for biological function and organism fitness. In contrast to thevast knowledge on the thermodynamics of protein stability in vitro, little is known about the factorsgoverning the kinetic stability that defines the lifetime of the native state of proteins within the cell.Here we show that the kinetic stability of the metallo-β-lactamase (MBL) NDM-1 in the bacterialperiplasm is optimized to withstand metal restriction or limitation at the host-pathogen interface.NDM-1 provides one of the most widespread mechanisms of carbapenem resistance in Gram negativebacteria. Despite the enhanced stability of NDM-1 in vitro, the non-metalated (apo) NDM-1 isrecognized and degraded by the periplasmic protease Prc. NMR experiments reveal that the flexibilityof the C-terminal domain of apo-NDM-1 permits degradation. Zn(II) binding renders the proteinrefractory to degradation by quenching this flexibility. In addition, apo-NDM-1 is anchored to the outermembrane, a localization that makes it less accessible to Prc and less prone to aggregate. Membraneanchoring also protects apo-NDM-1 from DegP, a quality control protease which degrades misfolded,non-metalated NDM-1 precursors. More recent clinical variants of NDM accumulate substitutions atthe C-terminus that quench its flexibility, therefore enhancing their stability towards proteolysis.These interrelated observations provide direct evidence of how the kinetic stability of a protein isoptimized within the bacterial cell, and links MBL-mediated resistance with essential cellularmetabolism within the bacterial periplasm. More generally, these processes also reveal thatunderstanding protein homeostasis in the cell is fundamental to comprehending protein evolution.