Stepwise order in protein complex assembly: approaches and emerging themes

Abstract

Protein-based nanomachines drive every cellular process. An explosion of high-resolution structures of multiprotein complexes has improved our understanding of what these machines look like and how they work, but we still know relatively little about how they assemble in living cells. For example, it has only recently been appreciated that many complexes assemble co-translationally, with at least one subunit still undergoing active translation while already interacting with other subunits. One aspect that is particularly understudied is assembly order, the idea that there is a stepwise order to the subunit-subunit associations that underlies the efficient assembly of the quaternary structure. Here, we integrate a review of the methodological approaches commonly used to query assembly order within a discussion of studies of the 20S proteasome core particle, septin protein complexes, and the histone octamer. We highlight shared and distinct properties of these complexes that illustrate general themes applicable to most other multisubunit assemblies.

Keywords: assembly; oligomerization; order; protein complexes; subunits.

Figure 1.

General principles governing assembly order. (A) An assembly chaperone (blue) transiently occupies a subunit–subunit interaction interface. (B) Interaction between two subunits alters the conformation of another interface, allowing a subsequent assembly step. (C) Co-translational interactions between subunits can drive assembly order if distinct assembly interfaces are distantly located in a nascent polypeptide. (D) Subunit compartmentalization, such as primarily nuclear localization of one subunit, can drive assembly order, especially if other steps occur in the cytoplasm during or soon after synthesis.

Figure 2.

Stepwise pathway of 20S proteasome core particle assembly. (A) A simplified pathway of proteasome core particle assembly showing stepwise addition of a few β subunits onto a pre-assembled ⍺ ring. The complete ⍺ ring is shown at left with associated assembly chaperones hidden. The assembly intermediate in the top centre shows the chaperone PAC3–PAC4 bound mostly to the ⍺5 subunit, plus addition of β1 and β2. At right, addition of β3 is accompanied by eviction of PAC3–PAC4. At bottom, a complete ⍺ and β ring. Protein structures are from PDB 8QYJ, 8QYO and 8QYS and do not necessarily reflect the conformations seen in each solved structure [6]; in some cases, subunits not shown in a complex were present in the solved structures but were simply hidden for this figure. (B) Conformational changes in the β2 subunit during steps along the CP pathway as determined by cryoEM (PDB structures 8QYJ, 8QYM, 8QYS, 8QYO) [6]. ‘Missing’ regions of the protein reflect proteolysis, or poorly structured regions that were not assigned locations.

Figure 3.

Stepwise pathways for septin complex assembly. (A) Left, illustration of pairwise interactions between purified individual budding yeast septins [27], with curved arrows indicating homotypic interactions. Middle, the early model for budding yeast septin organization within hetero-oligomeric complexes. Right, the actual organization of septin hetero-oligomers, including a representation of the structure of an actual hetero-octamer based on PDB 8PFH [28]. (B) Stepwise assembly pathways of budding yeast septin hetero-octamers, as determined by CSD-BiFC [14]. Cartoons represent septin subunits as in (A). The red ‘X’ and green check marks illustrate allosteric conformational changes that ‘activate’ a specific interaction interface. (C) Two alternative septin assembly pathways are active in organisms in which the purple subunit is able to hydrolyse GTP to GDP, driven by distinct affinities of the GTP- versus GDP-bound conformations of the purple subunit for (left) the pink subunit versus (right) another GDP-bound molecule of the purple subunit [29]. Portions of this figure were adapted from [14] under license CC BY 4.0.

Figure 4.

Stepwise pathways for histone octamer/nucleosome assembly. A simplified illustration of the presumptive pathway of histone octamer/nucleosome assembly. Nascent histone monomers are engaged in the cytoplasm by general chaperones (grey circles) before heterodimers are bound by histone-specific assembly chaperones (NAP1 and ASF1) which recruits karyopherins/importins that drive nuclear import. There, H3–H4 dimers are handed off to CAF-1, which brings two heterodimers together with DNA to form a DNA-associated tetramer. NAP1 facilitates incorporation of H2A–H2B dimers into octamers/nucleosomes. Protein structures were based on PDBs 5G2E [45], 1ID3 [46], 8J6T [47] and 2HUE [48].