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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.

SUBMITTER: Ross J

PROVIDER: S-EPMC8791618 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.

Ross Jennifer J McIver Zak Z Lambert Thomas T Piergentili Cecilia C Bird Jasmine Emma JE Gallagher Kelly J KJ Cruickshank Faye L FL James Patrick P Zarazúa-Arvizu Efrain E Horsfall Louise E LE Waldron Kevin J KJ Wilson Marcus D MD Mackay C Logan CL Baslé Arnaud A Clarke David J DJ Marles-Wright Jon J

Science advances 20220126 4

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant <i>Haliangium ochraceum</i> 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 ...[more]

PMID: 35080974

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Project description:Disaggregation and microtubule-severing nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily assemble into ring-shaped hexamers that enable protein remodeling by coupling large-scale conformational changes with application of mechanical forces within a central pore by loops protruding within the pore. We probed the asymmetric pore motions and intraring interactions that support them by performing extensive molecular dynamics simulations of single-ring severing proteins and the double-ring disaggregase ClpB. Simulations reveal that dynamic stability of hexameric pores of severing proteins and of the nucleotide-binding domain 1 (NBD1) ring of ClpB, which belong to the same clade, involves a network of salt bridges that connect conserved motifs of central pore loops. Clustering analysis of ClpB highlights correlated motions of domains of neighboring protomers supporting strong interprotomer collaboration. Severing proteins have weaker interprotomer coupling and stronger intraprotomer stabilization through salt bridges involving pore loops. Distinct mechanisms are identified in the NBD2 ring of ClpB involving weaker interprotomer coupling through salt bridges formed by noncanonical loops and stronger intraprotomer coupling. Analysis of collective motions of PL1 loops indicates that the largest amplitude motions in the spiral complex of spastin and ClpB involve axial excursions of the loops, whereas for katanin they involve opening and closing of the central pore. All three motors execute primarily axial excursions in the ring complex. These results suggest distinct substrate processing mechanisms of remodeling and translocation by ClpB and spastin compared to katanin, thus providing dynamic support for the differential action of the two severing proteins. Relaxation dynamics of the distance between the PL1 loops and the center of mass of protomers reveals observation-time-dependent dynamics, leading to predicted relaxation times of tens to hundreds of microseconds on millisecond experimental timescales. For ClpB, the predicted relaxation time is in excellent agreement with the extracted time from smFRET experiments.

Dataset Information

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.

Publications

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.

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