Petrovicet al., Science 376 , eabm9798 (2022) 10 June 2022 5of18
Fig. 3. Structural and biochemical analyses of the Nup192-Nup145N inter-
action.(A) Domain structures ofC. thermophilumNup192, Nic96, Nup53,
and Nup145N and the effect of each five-alanine substitution on Nup145N binding
to Nup192, as assessed by SEC and indicated by colored boxes above the
Nup145N primary sequence. (B) Summary of SEC binding analysis identifying
the minimal Nup145NR1(red) region sufficient for Nup192 binding. +++, no effect;
++, weak effect; +, moderate effect;–, abolished binding. (C to E) Cartoon
representation of (C) the 3.2-ÅC. thermophilumNup192•Nic96R2•Nup53R1•Nup145NR1
single-particle cryo-EM structure. Insets indicate regions magnified to illustrate
molecular details of (D) the Nup192-Nup53R1and (E) the Nup192-Nup145NR1
interactions. Red circles indicate residues involved in the Nup192-Nup53R1
( 41 ) and Nup192-Nup145NR1interactions. (F) Effect of Nup145N alanine
substitutions (cyan squares) on Nup192 binding, assayed by SEC (left). Effect
of structure-guided Nup192 alanine substitutions on SUMO-Nup145NR1binding,
assayed by SEC (right). (G) KDs determined by triplicate ITC experiments,
with the mean and associated standard error reported. (H) SEC-MALS analysis
of Nup192•SUMO-Nic96R2•Nup53•Nup145N and Nup192•SUMO-Nic96R2•
Nup53•SUMO-Nup145NR1complex formation and disruption by mutants.
Measured molecular masses are indicated, with respective theoretical masses
provided in parentheses.RESEARCH | STRUCTURE OF THE NUCLEAR PORE