Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment

Abstract

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo–electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

How Pore Architecture Regulates the Function of Nanoscale Protein Compartments

“…Apart from carboxysomes, a pivoting motion across a hinge region in the presence of a potential hydrogen bonding network was also postulated for pore gating at five-fold axes of symmetry in the H. ochraeum encapsulin. 89 This structural transition is similar to the aforementioned pivoting observed in the HIV-1 capsid, but whether it occurs in response to a molecular trigger requires further exploration. In general, more structural and functional studies are required to better understand these gating strategies and their role in regulating permeability.…”

“…A particularly intriguing aspect of protein compartment porosity is the flexibility and conformational changes that can occur at pores. Several studies have alluded to pore conformational changes using either molecular dynamics simulations, or structural or functional studies. , What remains to be uncovered is a dynamic model describing such conformational changes and their triggers in the context of either native or engineered functions of a protein compartment. This would consolidate our current model for selective pore permeability, which is largely based on static pore size and electrostatics.…”

“…The free hexamers have been thought to act as dynamic “caps” for facet-embedded hetero-hexameric pores following specific physicochemical triggers (Figure b). Apart from carboxysomes, a pivoting motion across a hinge region in the presence of a potential hydrogen bonding network was also postulated for pore gating at five-fold axes of symmetry in the H. ochraeum encapsulin . This structural transition is similar to the aforementioned pivoting observed in the HIV-1 capsid, but whether it occurs in response to a molecular trigger requires further exploration.…”

Engineered Protein Nanocages for Concurrent RNA and Protein Packaging In Vivo

“…Among naturally occurring protein nanocages, encapsulins have emerged as an alternative and attractive engineering platform for applications in medicine, catalysis, and nanotechnology. − Encapsulins are self-assembling icosahedral protein compartments composed of a single type of shell protomer possessing the HK97 phage-like fold. − They can assemble into T1 (60 subunits, ca. 24 nm), − T3 (180 subunits, ca. 32 nm), , and T4 (240 subunits, ca.…”

Equilibrium dynamics of a biomolecular complex analyzed at single-amino acid resolution by cryo-electron microscopy

“…For example, capsid “breathing” allows the externalization of certain CP regions that carry signals required for infection by some viruses (Bothner, Dong, Bibbs, Johnson, & Siuzdak, 1998; Bothner & Hilmer, 2011; Bothner et al, 1999; Lewis, Bothner, Smith, & Siuzdak, 1998; Roivainen, Piirainen, Rysa, Narvanen, & Hovi, 1993). Conformational fluctuation at equilibrium is a biologically critical feature also of many other biomolecular machines (Agarwal, Bernard, Bafna, & Doucet, 2020; DuBay, Bowman, & Geissler, 2015; Hodge, Benhaim, & Lee, 2020; Ross et al, 2022).…”