Cryo-EM structure of a thermostable bacterial nanocompartment.

Timothy Wiryaman,Navtej Toor

doi:10.1107/s2052252521001949

Timothy Wiryaman, Navtej Toor

Open Access

https://doi.org/10.1107/s2052252521001949

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Journal: IUCrJ	Publication Date: Apr 2, 2021
Citations: 15	License type: CC BY 4.0

Affiliation: University of California, San Diego

Abstract

Protein nanocompartments are widespread in bacteria and archaea, but their functions are not yet well understood. Here, the cryo-EM structure of a nanocompartment from the thermophilic bacterium Thermotoga maritima is reported at 2.0 Å resolution. The high resolution of this structure shows that interactions in the E-loop domain may be important for the thermostability of the nanocompartment assembly. Also, the channels at the fivefold axis, threefold axis and dimer interface are assessed for their ability to transport iron. Finally, an unexpected flavin ligand was identified on the exterior of the shell, indicating that this nanocompartment may also play a direct role in iron metabolism.

Highlights

Many species of bacteria and archaea have nanocompartments, which are protein assemblies that serve as nonmembranous compartments (Nichols et al, 2017; Giessen & Silver, 2017)
One major difference between encapsulins and viral capsids is that bacterial nanocompartments have binding sites in the interior for a specific peptide tag found on the C-terminus of cargo proteins (Sutter et al, 2008; Cassidy-Amstutz et al, 2016)
A codon-optimized gene coding for T. maritima encapsulin (TmEnc) was inserted into pET-11a and transformed into Escherichia coli Rosetta2 (DE3) cells (Novagen). 2 ml lysogeny broth with 100 mg mlÀ1 carbenicillin and 25 mg mlÀ1 chloramphenicol was inoculated with the strain expressing TmEnc and grown to an OD600 of 0.6 at 37C in a rotary shaker. 1 l of autoinduction medium with carbenicillin and chloramphenicol was inoculated with 1 ml of the starter culture, incubated at 37C to an OD600 of 2.9 with shaking and transferred to 20C for overnight protein expression

Summary

Introduction

Many species of bacteria and archaea have nanocompartments, which are protein assemblies that serve as nonmembranous compartments (Nichols et al, 2017; Giessen & Silver, 2017) These bacterial nanocompartments, which are known as encapsulins, strongly resemble icosahedral viral capsids in structure, there is little sequence conservation between these complexes (Sutter et al, 2008). One major difference between encapsulins and viral capsids is that bacterial nanocompartments have binding sites in the interior for a specific peptide tag found on the C-terminus of cargo proteins (Sutter et al, 2008; Cassidy-Amstutz et al, 2016) These cargo proteins generally catalyze reactions that remove toxic metabolites or reactions involving toxic substrates, intermediates or products (Giessen & Silver, 2017). The cargo proteins are diverse and include dye-decolorizing peroxidases (DyPs), ferritin-like proteins, iron-mineralizing encapsulin-associated firmicute (IMEF) proteins, hemerythrins, copper nitrite reductase/ hydroxylamine oxidoreductase fusion proteins, cysteine desulfurases and polyprenyl synthetases (Giessen & Silver, 2017; Nichols et al, 2020; Tracey et al, 2019)

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