Abstract
Encapsulin is a class of nanocompartments that is unique in bacteria and archaea to confine enzymatic activities and sequester toxic reaction products. Here we present a 2.87 Å resolution cryo-EM structure of Thermotoga maritima encapsulin with heterologous protein complex loaded. It is the first successful case of expressing encapsulin and heterologous cargo protein in the insect cell system. Although we failed to reconstruct the cargo protein complex structure due to the signal interference of the capsid shell, we were able to observe some unique features of the cargo-loaded encapsulin shell, for example, an extra density at the fivefold pore that has not been reported before. These results would lead to a more complete understanding of the encapsulin cargo assembly process of T. maritima.
Highlights
Membrane-based organelles in eukaryotic cells are important cellular structures with various functions, including confining enzymes and substrates to enhance enzymatic activity and limit the propagation of potential damage caused by toxic products
It indicates a successful assembly of the heterologous IDM complex inside of the encapsulin shell with the insect cell expression system
We are presenting the first case of insect cell expression of encapsulin
Summary
Membrane-based organelles in eukaryotic cells are important cellular structures with various functions, including confining enzymes and substrates to enhance enzymatic activity and limit the propagation of potential damage caused by toxic products. Compartmentation is realized by protein-based containers [1,2]. Encapsulin is a kind of protein compartment that exists in a wide variety of bacteria and archaea [3,4,5]. Encapsulins are assembled by capsid proteins into an icosahedral shell with a diameter between 24 and 42 nm, which encapsulates native cargo proteins involved in oxidative stress and iron mineralization [3]. The conserved C-terminal extension of native cargo proteins, which is called cargo-loading peptide (CLP), mediates the interaction between the interior surface of encapsulin and cargo protein [6]. There are several examples of non-native cargo by fusing the CLP to heterologous proteins like teal fluorescent protein, superfolder green fluorescent protein (sfGFP), and mNeonGreen in Brevibacterium linens, T. maritima, and Myxococcus xanthus, respectively [7,8,9]. The conformational changes and the packaging mechanisms of encapsulin and its cargos are poorly understood
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