Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Argentina.
Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
Nat Chem Biol. 2023 Sep;19(9):1116-1126. doi: 10.1038/s41589-023-01319-0. Epub 2023 May 15.
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.
蛋白质稳定性是其发挥生物学功能的必要属性。与体外蛋白质稳定性的大量知识形成鲜明对比的是，人们对影响细胞内稳定性的因素知之甚少。在这里，我们发现金属-β-内酰胺酶（MBL）新德里 MBL-1（NDM-1）在金属限制下是一种动力学不稳定的蛋白质，它通过获得不同的生化特性来进化，从而优化其细胞内稳定性。非金属化（脱辅基）的 NDM-1 会被周质蛋白酶 Prc 降解，该酶识别其部分无结构的 C 末端结构域。Zn(II)结合使该蛋白不易被降解，因为它使该区域的柔韧性降低。膜锚定使脱辅基的 NDM-1 更难被 Prc 识别，并保护其免受 DegP（一种细胞蛋白酶，可降解错误折叠的非金属化 NDM-1 前体）的降解。NDM 变体在 C 末端积累了使它们的柔韧性降低的取代，从而增强了它们的动力学稳定性并绕过了蛋白水解。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，突出了细胞内蛋白质平衡的重要性。
