Sequence space of Xpo1-dependent NESs reveals a functional affinity ceiling

Exportin 1 (Xpo1/Crm1) exports hundreds of proteins from the nucleus to the cytoplasm. It recognizes nuclear export signals (NESs) with 4-5 hydrophobic {Phi} residues separated by spacer residues. Here we explored the sequence space of the most common NES class by a high-throughput ratiometric scoring of Xpo1-binding, combining phage display with deep sequencing. This provided a positional preference map and revealed that not only the {Phi}-positions but also spacer and flanking residues are critical for NES activity. We validated these data in vivo and with a new equilibrium affinity measurement that exploits the competition for Xpo1 when NES{middle dot}Xpo1{middle dot}RanGTP complexes partition into an FG phase. Guided by these preferences, we designed peptides that satisfy the established NES consensus but fail to confer export. Conversely, we engineered NESs that bind Xpo1 with low picomolar affinity -- explained by a crystal structure. Such extreme binders, however, are no longer released from Xpo1 and block export in trans, explaining why natural NESs remain modest in affinity. Our data provide a framework for predicting, identifying, and engineering NESs and other peptide-based signals.