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-β-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.

蛋白质稳定性是生物功能的重要特性。与体外蛋白质稳定性的大量知识相比，我们对细胞内稳定性的控制因素知之甚少。在这里，我们证明金属-β-内酰胺酶 (MBL) New Delhi MBL-1 (NDM-1) 是一种受金属限制的动力学不稳定蛋白，它通过获得不同的生化特征来优化其细胞内稳定性而进化。非金属化 (apo) NDM-1 被周质蛋白酶 Prc 降解，Prc 识别其部分非结构化的 C 端结构域。Zn(II) 结合通过淬灭该区域的灵活性而使蛋白质难以降解。膜锚定使 apo-NDM-1 不易接触到 Prc，并保护其免受 DegP 的影响，DegP 是一种细胞蛋白酶，可降解错误折叠的非金属化 NDM-1 前体。NDM 变体在 C 末端积累取代，从而淬灭其灵活性，增强其动力学稳定性并绕过蛋白水解。这些观察结果将 MBL 介导的耐药性与必要的周质代谢联系起来，强调了细胞蛋白质稳态的重要性。