Pore structure controls stability and molecular flux in engineered protein cages

Abstract

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. Pores lined with anionic residues resulted in more robust assemblies than their corresponding cationic variants. We then determined seven cryo-EM structures of pore variants at resolutions between 2.5-3.6 Å. Together with stopped-flow kinetics experiments for quantifying cation influx, we uncover the complex interplay between pore size, charge, and flexibility that controls molecular flux in protein cages, providing guidance for future nanoreactor designs.Abstract Figure

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment

“…This observation has important implications for efforts to engineer the pores of encapsulin nanocages. Where early efforts to widen the five-fold pores have demonstrated an increase in mass-transport of model substrates across the encapsulin shell (26), more recent investigations into pore modifications have shown that the shell does not act as a strong barrier to the passage of the small lanthanide substrates tested (27). Our results provide an explanation for these observations, where a dynamic and flexible pore would not act as a barrier to the passage of small ligands, such as divalent cations, across the shell.…”

Nanotechnological Applications Based on Bacterial Encapsulins

“…Pores in the nanocompartment regulate the molecular flux through the shell by size and by charge [6,53,81,82], and constitute one of the key parameters in controlling encapsulated protein activity [83]. Their role as a molecular sieve is thought to assist the flux of correct substrates [36,49], preventing undesired reactions.…”

“…Encapsulins are usually further purified by SEC or ion-exchange chromatography, with a final SEC polishing step. For some applications, high resolution SEC has been deemed essential [82].…”

“…The high catalytical activity of encapsulated enzymes requires adaptation between pore characteristics and metabolite size and charge [63,83]. Two studies analyzed the influence of pore size and charge on mass transport [81,82] by systematically mutating the loop region that delineates the pores at the fivefold axis of the EncTm nanocompartment. They generated structural libraries of nanocompartments with pore variants and analyzed the effect of pore size and charge on nanocompartment structure and on small molecule flux through the nanocompartment shell.…”

Triggered reversible disassembly of an engineered protein nanocage

“…Encapsulin nanocompartments have recently emerged as a particularly versatile bioengineering tool, resulting in their application as bionanoreactors, targeted delivery systems, and nano-and biomaterials production platforms. [8][9][10][11] Encapsulins are icosahedral protein nanocages found in bacteria and archaea with triangulation numbers of T=1 (24 nm), T=3 (32 nm) or T=4 (42 nm) containing sub-nanometer pores at the symmetry axes. 12 They self-assemble from a single HK97-fold capsid protein into 60mer (T=1), 180mer (T=3) or 240mer (T=4) protein cages and are involved in oxidative stress resistance, [13][14][15][16] iron mineralization and storage, 17,18 and sulfur metabolism.…”