Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage

Abstract

Encapsulins are microbial protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. Due to their favorable properties, including high thermostability, protease resistance, and robust heterologous expression, encapsulins have become popular bioengineering tools for applications in medicine, catalysis, and nanotechnology. Resistance against physicochemical extremes like high temperature and low pH is a highly desirable feature for many biotechnological applications. However, no systematic search for 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 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. Our results expand the biotechnological application range of encapsulin nanocages toward potential uses under strongly acidic conditions and highlight pH-responsive encapsulin pore dynamics.

The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell

“…N-arms of Family 2B encapsulins display high sequence and length variability, which may allow different alternate orientations of the N-arms and consequently different two-fold pore states which may be static or dynamic 22 . Dynamic five-fold pores have recently been reported in Family 1 encapsulins 74-76 . Furthermore, as mentioned above, some Family 2B gene clusters encode for two distinct Family 2B encapsulin shell proteins, which may form heterogeneous two-component shells with a mixture of open and closed two-fold pores, depending on the respective N-arm states of the constituent shell protomers.…”

Structural basis for peroxidase encapsulation in a protein nanocompartment

“…The observed pore sizes and charges are in principle compatible with the transmission of a wide range of small molecules and do not meaningfully narrow down the range of potential native DyP substrates. This is particularly true when considering the fact that some Family 1 encapsulins have recently been reported to possess dynamic pores able to open and close based on environmental conditions and potentially other so far unknown stimuli 54,55 . Even though icosahedral (I) refinement yielded high quality cryo-EM density for the encapsulin shell, DyP cargo could not be visualized because of compositional heterogeneity and conformational flexibility.…”

Trendbericht Biochemie 2023 (2/3): Encapsulin in der Katalyse

“…1,9,12,13) Natürliche Gastproteine von Encapsulin wie die Entfärbungsperoxidase DyP oder die Hydroxylaminoxidoreduktase HAO sind ähnlich aktiv wie freie Enzyme, aber thermisch stabiler und basen-oder säureresistenter. 3,12,16,17)…”