Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage

Abstract

Encapsulins are protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. They are found in a wide variety of bacteria and archaea, including many extremophiles, and are involved in iron and sulfur homeostasis, oxidative stress resistance, and secondary metabolite production. Resistance against physicochemical extremes like high temperature and low pH is a key adaptation of many extremophiles and also represents a highly desirable feature for many biotechnological applications. However, no systematic characterization of acid stable encapsulins has been carried out, while the influence of pH on encapsulin shells has so far not been thoroughly explored. Here, we report on a newly identified encapsulin nanocage (AaEnc) from the acid-tolerant bacterium Acidipropionibacterium acidipropionici. Using transmission electron microscopy, dynamic light scattering, and proteolytic assays, we demonstrate its extreme acid tolerance and resilience against proteases. We structurally characterize the novel nanocage using cryo-electron microscopy, revealing a dynamic five-fold pore that displays distinct "closed" and "open" states at neutral pH, but only a singular "closed" state under strongly acidic conditions. Further, the "open" state exhibits the largest pore in an encapsulin shell reported to date. Non-native protein encapsulation capabilities are demonstrated, and the influence of external pH on internalized cargo is explored. AaEnc is the first characterized highly acid stable encapsulin with a unique pH-dependent dynamic pore and its molecular characterization provides novel mechanistic details underlying the pH stability of large dynamic protein complexes.