We have presented 3D structures of an entire NPC subcomplex, the heptameric Nup84 complex from budding yeast. Our data confirm the overall architecture that was proposed previously based on 2D EM16
. Averaging of images allowed us to discern additional details, such as four globular regions and the asymmetry of the two arms of the particle, and to characterize the conformational heterogeneity of the particle. The 3D maps, in combination with protein labeling experiments, enabled us to dock available nup crystal structures into the heptamer structure.
The EM structures () do not necessarily represent the conformation of the heptameric complex in the context of the NPC since (i) interactions with other nups may affect the conformation of the heptameric complex, and (ii) the present structures are of the particle bound to a planar support film, whereas in the context of the NPC, the heptameric complex coats a highly curved surface. Distortions of the particle structure caused by negative staining and by missing-cone effects due to incomplete angular coverage of particle views are a potential concern, but they are unlikely to be dramatic in the present study, since the particle is not very extended in the direction perpendicular to the carbon support film. The simultaneous iterative reconstruction technique was used to minimize missing-cone effects. The docking of nups into the EM map () represents the best possible fit given the current data; future higher-resolution EM maps and additional crystal structures may lead to a refinement of nup positions and orientations.
Despite these caveats, the present 3D structures yield fundamental insights into the architecture of the heptameric complex. The main architectural principle of the heptameric complex is that the globular domains at the ends of the arms and the stem are formed by β-propeller domains, whereas the thinner connecting segments are formed mainly by α-solenoid folds. While the crystal structure of Nup120 is not yet available, we expect Nup120 to conform to this principle: the shape of the long arm strongly suggests that the predicted Nup120 β-propeller localizes to the thick, globular end of the arm, whereas the predicted α-helical regions form the thinner connection to the vertex. This arrangement is supported by the 2D class averages (): the long arm ends in a round shape ~5 nm in diameter with a central hole or depression, compatible with a β-propeller in top view. Intriguingly, the same architectural principle of α-solenoid arms ending in β-propeller domains is also found in the clathrin triskelion31
, thus lending further support to the hypothetical evolutionary relationship between vesicle coats and the heptameric complex.
β-propellers occur in many biological contexts, frequently acting as platforms for interactions with other proteins. The structural basis for this function is their rigid fold and the availability of several highly variable interaction surfaces.32
Remarkably, the surfaces of the four β-propellers in the heptamer are mostly exposed, and thus available for interactions with other proteins. As suggested previously6,17,18
, the β-propellers may be involved in higher-order interactions between heptameric complexes within the NPC. Such an arrangement occurs in the COPII vesicle coat33
. Moreover, a scaffold formed by heptameric complexes is likely to form a platform that organizes other nups within the NPC.
Whereas the β-propellers are rigid structural units, the connecting regions formed by α-solenoids and, possibly, by unstructured regions are likely to account for the conformational flexibility of the heptameric complex. Flexibility of α-solenoid arms was described for both COPII coatamers33
and clathrin triskelia34
, where it is thought to allow the formation of vesicle coats in different sizes.
The flexibility of the heptameric complex is also potentially of physiological relevance. Flexibility of the entire NPC was described2,3,35
and may reflect conformational changes that accompany active transport. In particular, dilation of the NPC may be required to allow passage of large cargoes, such as ribosomal subunits. Molecular sliding of nups located near the central channel of the NPC was suggested to form the basis for NPC dilation4
. It is likely that conformational changes of these central nups would occur in concert with conformational changes of the more peripheral nups, including the heptameric complex. A further requirement for flexibility may apply to the vertebrate homologue of the heptameric subcomplex, which has additional functions outside the NPC during mitosis1
, and may adopt distinct conformations in different cellular contexts.
The heptameric complex was reported to play an essential role in the formation of NPCs, both post-mitotically and during interphase. Immunodepletion of the vertebrate homologue of the heptamer from nuclear assembly reactions leads to the formation of a continuous nuclear envelope devoid of pores10,11
. Similarly, the heptamer is required for de novo
insertion of NPCs into the interphase nuclear envelope12
. While the mechanism of NPC assembly is currently unknown, a specific structural role for the heptameric complex in this process can be envisaged based on its structure and its affinity for highly curved membrane surfaces.
Formation of new NPCs during interphase requires the formation of a fusion pore between the outer and inner nuclear membranes. In other biological contexts, membrane fusion was shown to occur by a stepwise process: apposition of two membranes, hemifusion between the inner leaflets of the two lipid bilayers, reversible formation of a small fusion pore, stabilization and expansion of the fusion pore36
. In different biological processes, such as exocytosis or viral membrane fusion, these steps are catalyzed by specific proteins that interact with the membranes to overcome the inherent energy barriers of each step along the fusion pathway, and to control the geometry of fusion.
The heptameric complex may function in one or several phases during the formation of nuclear envelope pores. The formation of the initial fusion pore is likely catalyzed by integral membrane proteins, possibly by the poms, which are components of the mature NPC. The heptamer may then stabilize initial fusion pores, by binding to the sharply bent membrane lining the pore. The following step of fusion pore expansion is particularly interesting in the case of nuclear envelope pores: whereas in other biological contexts, such as vesicle fusion, fusion pores expand maximally to integrate the vesicle membrane into the target membrane, the nuclear envelope fusion pore expands to a defined diameter of ~100 nm to accommodate the NPC. A scaffold formed by several heptamers may control the final size of the fusion pore, and thus act as a molecular ruler. An octagon with a diameter of 100 nm has edges that are ~38 nm long. Such an octagon could be formed by a previously suggested18
head-to-tail arrangement of eight heptameric complexes in a ring: The heptameric complex is ~45 nm long (), allowing for some overlap at the head-to-tail interface.
Once the heptamer scaffold has stabilized the 100 nm pore, it can serve as a platform for the recruitment of other nups. The eightfold symmetry of the NPC may be dictated by the eightfold symmetry of the initial scaffold formed by heptameric complexes. Interestingly, the length of the heptameric complex was constant in the different conformations we observed (). This means that a ring of heptamers could maintain a fixed size based on head-to-tail interactions, while the flexibility of the heptamer would buffer conformational changes of other parts of the NPC that are anchored to the heptamer ring.