The discovery of distinct structures embedded in the nuclear envelope can be traced back to electron microscopic studies of thin sections of cells carried out in the 1950s, principally by Michael Watson working in the laboratory of Keith Porter and George Palade at Rockefeller University. These studies culminated in 1958 with the publication of a seminal paper, in which Michael Watson coined the term "nuclear pore complex" (NPC), implying that these large structures provide the principal route for molecular traffic into and out of the nucleus.
In 1986, our laboratory characterized the first proteins that make up the NPC. We termed these proteins "nucleoporins" (nups) and, to distinguish between nups, we post-scripted each nup with a number corresponding to its calculated molar mass in kilodaltons. In 2004, we began crystallographic analyses of predicted ordered regions of nups, with the long-term goal of piecing together NPC structure at atomic resolution.
Early on, we discovered that interacting segments of nups crystallized in distinct conformers, pointing to the dynamic nature of their interactions. Especially striking examples for alternative conformers were discovered for two nups, Nup58 and Nup54. Together with Nup62, these two nups had previously been localized by immunoelectron microscopy to the very center of the NPC. We suggested a model, based on several of our crystal snapshots of cognate segments of these three nucleoporins, for the structure and dynamics of a central channel of the NPC. This model displays two principal features: (1) a midplane ring and (2) triple helices (fingers) that are attached to the midplane ring and project in an alternate fashion to its nucleoplasmic and cytoplasmic side. For the dynamics of the midplane ring, we postulated a “ring cycle”, in which conformers of cognate segments of Nup58 and Nup54 reversibly transit from a heterododecamer to three homotetramers. Based on the established eightfold rotational symmetry of the NPC, as well as on the dimensions of the conformers, eight heterododecamers form a single large ring of ~40 nm diameter, whereas eight homotetramers each form three smaller rings of ~10–20 nm diameter. The large ring is the dilated (open) form, whereas the (stacked or tucked-in) smaller rings are the constricted (closed) form of the central channel. We further proposed that, akin to ligand-gated ion channels, dilation and constriction of the midplane ring are regulated by transport factors functioning as ligands. That is, in the specific example of the Nup58•Nup54 midplane ring, binding of transport factor to the Phe-Gly (FG) repeat region of Nup58 would stabilize the dilated conformer of the midplane ring.
The predictions of this gating mechanism were borne out by biophysical experiments. Specifically, we showed that transport factor binding to the FG region of Nup58 allosterically affects its upstream region to enhance binding of Nup58 to Nup54. Hence, binding of transport factor stabilizes the dilated conformer (heterododecamer) at the expense of the constricted conformer (homotetramers) (see visualization of ring cycle and stabilization of dilated conformer by transport factor ligand. Allosteric sensing of transport factor occupancy is an unexpected and novel function for the FG region of a nup).
Our crystal structure-based model indicated that the central channel of a single NPC is composed of 128 molecules of Nup62, 64 molecules of Nup54, and 32 molecules of Nup58, collectively amounting to a mass of 12.3 million daltons, i.e., ~10 percent of the estimated mass of ~120 million daltons for a single NPC.
Presently, we are examining how hitherto "unsaturated" segments of the ordered region of Nup62 and Nup58 interact with surrounding nups. We are also testing our proposal that binding of transport factor to some of the nups surrounding the central channel may stabilize conformers that are compatible with the dilated state of the midplane ring. Such multiple binding reactions may cooperatively establish a dilated state of the entire NPC that can be regulated by transport factors from either its nucleoplasmic or cytoplasmic side.
As of March 11, 2016