Abstract 2079 Assembly dynamics of a protein nanocompartment with a novel two-component icosahedral architecture

Encapsulin nanocompartments are a class of prokaryotic organelles that enable specialized biochemistry through the sequestration of enzymatic cargo within a selectively permeable protein shell. Although encapsulin systems utilize a variety of enzymatic cargos, the encapsulin shell has only ever been shown to be composed of a single type of shell protomer. Here, we report the structural characterization and proposed assembly dynamics of an encapsulin nanocompartment with a novel two-component shell from Streptomyces lydicus. Separate heterologous expression and purification of the two encapsulin shell genes from the S. lydicus encapsulin operon revealed that only one of the proteins can assemble into icosahedral T=1 encapsulin shells. However, co-expression of the two genes demonstrated not only that the proteins will co-assemble to form chimeric encapsulin shells, but that encapsulin particles can flexibly incorporate varying amounts of both proteins. Furthermore, dynamic light scattering (DLS) and thermal ramp data for mixed shell samples suggest that there is an assembly preference for chimeric shells, resulting in greater shell stability. Using cryo-electron microscopy (cryo-EM), we determined the structures of the two different shell protomers, both containing the signature HK97-like protein fold utilized by all encapsulin shells to date, as well as an external domain with significant structural similarity to cyclic nucleotide (cNMP)-binding domains. To better understand the drivers of chimeric shell assembly, we used crosslinking mass spectrometry and computational approaches to determine possible protein-protein interactions within mixed shells. Our results suggest that the assembly of the two-component shell is likely random, yielding stochastically tiled mixed shells. Altogether, we present a structural characterization of the first known protein compartment to preferentially utilize two distinct protein components with the HK97-like protein fold. This work was supported by the National Science Foundation Graduate Research Fellowship Program (DGE 1841052) and the National Institutes of Health (R35GM133325).