the GDP-bound form of Ran from the GTP-bound species. Indeed, they bind RanGTP with a
≈1000 times higher affinity than RanGDP, which makes them efficient sensors of the RanGTP gradient. NTRs do not directly sense the nucleotide, but instead, very elegantly probe those regions of Ran that differ most between the nucleotide states – the switch loops I and II. Ran's C-terminal switch does not contribute to NTR binding, but plays a key role in the disassembly of NTR·RanGTP complexes (see Chapter 5). NTRs also contact Ran at several "invariant" loops and features of its "back side" (including the "basic patch", see below).
The comparison of the RanGTP complexes of Impβ, Transportin and CAS (Figure 1-5) reveals that all these NTRs enwrap Ran with their N-terminal arches (Vetter et al., 1999a;
Chook and Blobel, 1999; Matsuura and Stewart, 2004; Lee et al., 2005). In all cases, three distinct HEAT repeat regions contribute. The receptors' N-termini (which are most conserved among NTRs; Görlich et al., 1997; Fornerod et al., 1997b; Petosa et al., 2004) constitute the first Ran-binding region. This area interacts with switch II and also contacts α3 on the "back"
of Ran (see Figure 1-4). It is also near switch I, and for Transportin and CAS, there are indeed some relevant contacts. Region 2 extends over Ran's back and shields, among others, the "basic patch" of Ran. In RanGDP, large parts of this contacted area are held in check by Ran's C-terminal switch (Figure 1-4) and would hence be inaccessible for a transport receptor. The third region binds those loops of Ran that are involved in holding the nucleotide's guanine base (Figure 1-4a). Most importantly, however, Impβ also contacts switch I via this area. This interaction has not been described for Transportin, but slight conformational changes of the intra-HEAT 13 loop would be sufficient to establish such a contact. In the case of CAS, equivalent interactions are definitely absent, but here a very peculiar loop inserted into HEAT 19 contacts switch I. Another type of HEAT repeat insertion is noticeable from Figure 1-5 – the acidic insertions into HEATs 7 or 8. In Impβ and Transportin, they are part of contact area 2 and contribute to binding the "back" of Ran, including its "basic patch".
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Three complexes are drawn to scale and are shown in identical orientations with respect to Ran. NTRs are depiceted in gray, their helices are represented as cylinders. Those HEAT repeats that mediate interactions with Ran are numbered and colored in orange. Numbering is according to the specified references. Notable NTR regions are highlighted in magenta (acidic HEAT inserts) or green ("HEAT 19 insert" in CAS). Encircled numbers mark the respective Ran-binding regions. Ran is shown in light blue (tube representation) with switch I colored in red, switch II colored in cyan and the "basic patch" shown in dark blue.
(a) Kap95p (Impβ)·RanGTP complex (Lee et al., 2005; PDB-ID 2BKU).
(b) Transportin·RanGppNHp complex (Chook and Blobel, 1999, PDB-ID 1QBK).
(c) Kap60p (Impα)·Cse1p (CAS)·RanGTP complex (Matsuura and Stewart, 2004, PDB-ID 1WA5).
Kap60p (Impα) has been omitted for clarity.
N- and C-termini of NTRs are labeled. See text for further details.
For Transportin, these interactions are very elaborate and also involve Ran's guanine-binding loops. In CAS, the reported HEAT 8 insert does not contact Ran. In fact, the entire Ran-binding region 2 of CAS is not very pronounced, but here, also the cargo (which has been omitted in Figure 1-5c) contributes to the formation of the CAS·RanGTP complex. This cooperativity mechanism is discussed in Chapter 5.
22 arches for binding (Cingolani et al., 1999; Lee et al., 2003; Lee et al., 2006; Imasaki et al., 2007; Wohlwend et al., 2007; Mitrousis et al., 2008). This raises the question as to how Ran triggers efficient import cargo release. For Impβ two mechanisms have been proposed (Vetter et al., 1999a; Lee et al., 2005). The first mechanism involves a steric clash between Ran and cargo: the cargo- and Ran-binding sites partially overlap in the region that connects the two arches. For instance, both Ran and the IBB domain of Impα bind Impβ's acidic H8 loop.
However, this direct competition appears to be insufficient for productive disassembly of the import complex (Lee et al., 2005). A second, allosteric mechanism is required for complete release: the interactions of Ran with region 3 of Impβ (Figure 1-5a) increase the helicoidal pitch of the importin, which ultimately expels the IBB domain. Consistent with that (and quite impressively), the disruption of that region 3 interface (by point mutations in Ran) inhibits RanGTP-driven release of the IBB domain from Impβ but does not prevent RanGTP binding to the importin. For Transportin, the contacts of Ran with region 3 are less extensive, but here yet another allosteric cargo release strategy is employed, involving Transportin's strikingly long acidic H7 insert. In the cargo-bound state, the H7 insert appears to be disordered (Lee et al., 2006; Imasaki et al., 2007), but upon Ran binding, it is "forced" to a path that blocks Transportin's cargo-binding site in the C-terminal arch (Figure 1-5b, Chook and Blobel, 1999). In contrast to Impβ, cargo-bound and RanGTP-bound Transportin are virtually indistinguishable by their overall shapes.
How precisely Ran contacts exportins and thereby promotes export cargo loading was not understood at the beginning of my doctoral studies (with the exception of CAS, see Figure 1-5c and Chapter 5). The work presented in this thesis provides unprecedented insight into Ran-dependent CRM1 export complex formation. For the sake of simplicity, the role of Ran in nuclear export is discussed further in Chapter 5.
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