Table 1 Summary of ITC data.

Detailed description of rows 1 to 5 is provided in Materials and Methods. ∆G° and TS° were determined by ∆G° = −RT lnK and TΔS = ΔH° − ΔG°, where R and T are gas constant (1.99 cal mol−1 K−1) and temperature (288 K), respectively. The errors are the SDs obtained from the global fits, pertaining to uncertainties in both the model accuracy and measurement precision (i.e., model and experimental errors). (1) 53core dissociation profiles obtained at three protein concentrations were analyzed by a monomer-dimer equilibrium model. (2) 53C·Kap121 forward and reverse titrations, performed at various protein concentrations, were globally analyzed by a two-mode binding model, yielding thermodynamic constants for 1:1 and 2:1 (53C:Kap121) interactions in rows 2a and 2b. These parameters also describe interactions between monomeric 53core and Kap121. 53core·Kap121 forward and reverse titrations, performed at various protein concentrations, were globally analyzed by a multiple-equilibrium model using constants in rows 1, 2a, and 2b. The obtained parameters in 2c and 2d describe 2:1 and 2:2 interactions between 53core dimer and one and two molecules of Kap121, respectively. A2*B and A2B complexes differ in the oligomeric state of A (two monomers versus dimer). (3 and 4) Nic96·53core and Nup157·53core binding profiles were analyzed by a single-site 1:1 binding model. (5) Nic96 titrations into stoichiometric 53core:Kap121 or 53core:HsKapβ1 mixtures were analyzed by a single-site 1:1 model to obtain apparent K’s (Kapp) for 53core-Nic96 interactions in the presence of Kaps (rows 5a and 5a′). Titrations were globally analyzed by a multiple-equilibrium model to deconvolute Nic96 binding to monomeric and dimeric 53core·Kap121 complexes (rows 5b and 5c, respectively). Parameters describing Nic96 interactions with the monomeric 53core·Kap121 complex were independently obtained using a dimerization-deficient Nup53mut fragment (table S1 and fig. S7, A and B). The subscript i in Kap121i denotes the number of bound Kap molecules (i = 1 or 2).

NoteInteraction*KH° (kcal/mol)G° (kcal/mol)TS° (kcal/mol)
12A ↔ A21.0 (± 0.1) × 104 M−16.5 ± 0.2−5.3 ± 0.111.8 ± 0.2
2aA + B ↔ AB4.2 (± 0.7) × 106 M−1−2.7 ± 0.1−8.7 ± 0.16.0 ± 0.1
2b2A + B ↔ A2*B1.9 (± 0.5) × 1011 M−24.2 ± 0.7−14.9 ± 0.219.1 ± 0.8
2cA2 + B ↔ A2B5.6 (± 0.8) × 107 M−116.3 ± 1.4−10.2 ± 0.126.5 ± 1.4
2dA2 + 2B ↔ A2B23.9 (± 1.0) × 1014 M−2−1.3 ± 0.4−19.2 ± 0.117.9 ± 0.4
3A + D ↔ AD1.1 (± 0.3) × 106 M−1−10.1 ± 0.5−8.0 ± 0.2−2.1 ± 0.5
4A + C ↔ AC9.9 (± 0.9) × 105 M−1−4.4 ± 0.1−7.9 ± 0.13.5 ± 0.1
5a(AB)app + C ↔ (AB)appC1.3 (± 0.4) × 105 M−1−5.2 ± 0.6−6.7 ± 0.21.5 ± 0.6
5a′(AB′)app + C ↔ (AB′)appC1.0 (± 0.1) × 105 M−1−5.1 ± 0.4−6.6 ± 0.11.5 ± 0.4
5bAB + C ↔ ABC2.0 (± 0.9) × 105 M−1−6.7 ± 1.7−7.0 ± 0.30.3 ± 1.7
5cA2Bi + C ↔ A2BiC1.2 (± 0.2) × 105 M−1−2.5 ± 0.3−6.7 ± 0.14.2 ± 0.3

*53core (A), Kap121/HsKapβ1 (B/B′), Nic96 (C), and 157N (D).