Abstract
Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3. This pathway is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Here we identify the mechanisms of importin α3 selectivity for RCC1. We find this isoform binds RCC1 with one order of magnitude higher affinity than the generic importin α1, although the two isoforms share an identical NLS-binding groove. Importin α3 uses its greater conformational flexibility to wedge the RCC1 β-propeller flanking the NLS against its lateral surface, preventing steric clashes with its Armadillo-core. Removing the β-propeller, or inserting a linker between NLS and β-propeller, disrupts specificity for importin α3, demonstrating the structural context rather than NLS sequence determines selectivity for isoform 3. We propose importin α3 evolved to recognize topologically complex NLSs that lie next to bulky domains or are masked by quaternary structures.
Highlights
Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3
Using nano isothermal titration calorimetry (ITC), we established that isoform 3 has one order of magnitude higher binding affinity for RCC1 than importin α1 (KD = 0.62 ± 0.1 μM vs. 5.4 ± 0.3 μM) (Fig. 1a, b)
The β-propeller has a disk-like structure with two faces: one face that binds Ran[49] is solvent exposed, while the opposite face contacts the C-terminal portion of the tail, including the major NLS box, which is sandwiched between the importin α3 helical groove and the β-propeller (Fig. 2a)
Summary
Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3 This pathway is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. A growing number of specialized cellular NLS cargos like RCC124–27, STAT128, NF-kB29, 30 as well as viral factors influenza PB231 and Ebola VP2432 enter the nucleus in complex with specific importin α isoforms. These isoforms play an important role in cell differentiation[33], disease states, especially cancer[21] and viral infections[21, 34]. No of reflections (tot/unique) Rsym Rpim I/σI Completeness (%) Redundancy Refinement PDB ID Resolution (Å) No of reflections Rwork/Rfree* No of complexes in AU No of protein atoms Ramachandran Favored/allowed/outliers MolProbity Clashscore R.m.s deviations Bond lengths (Å) Bond angles (°)
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