Valadez, C. | Search bioRxiv

bioRxiv2026

Importins recognize the winged-helix fold of ETS transcription factors to mediate nuclear import

Protein trafficking between the cytoplasm and the nucleus is a fundamental process in eukaryotic cell biology. While linear nuclear localization signals (NLSs) are well-characterized, many nuclear proteins lack a predictable NLS. Here, we identify the ETS domain, a DNA-binding winged-helix fold, from ETS family transcription factors as a structure-encoded NLS. We show that ETS domains mediate nuclear import through direct recognition by multiple nuclear transport receptors, including IPO9. Cryo-electron microscopy analysis of the EHF:IPO9 complex reveals that the IPO9 wraps around the ETS domain and engages structural features throughout the winged-helix fold. Biochemical studies demonstrate that the ETS domain DNA-binding helix is critical for importin recognition and for NLS activity in mammalian cells. Comparison of IPO9 bound to EHF and the histone H2A:H2B dimer reveals distinct interaction hotspots, illustrating how IPO9 employs unique combinatorial binding surfaces to accommodate structurally diverse cargos. These findings define a new class of globular NLSs and highlight the adaptability of importins in recognizing distinct protein folds. Significance StatementNuclear import is essential for transcription factor function. However, many nuclear proteins lack recognizable nuclear localization signals (NLSs), leaving their trafficking mechanisms unresolved. Here, we identify the winged-helix DNA-binding domain of ETS transcription factors as a structure-encoded NLS shared across the ETS family of proteins. We show that multiple importins directly interact with this globular domain and define the molecular basis for cargo recognition by determining the cryo-EM structure of an importin bound to an ETS family protein. These studies establish a new class of globular NLSs and shed light on how individual importins can recognize diverse protein folds. We also provide mechanistic insight into nuclear trafficking defects that are caused by disease-linked ETS transcription factor mutations.

biochemistry↗

bioRxiv2024

Enigmatic missense mutations can cause disease via creation of de novo nuclear export signals

Disease-causing missense mutations that occur within structurally and functionally unannotated protein regions can guide researchers to new mechanisms of protein regulation and dysfunction. Here, we report that the thrombocytopenia-, myelodysplastic syndromes-, and leukemia-associated P214L mutation in the transcriptional regulator ETV6 creates an XPO1-dependent nuclear export signal to cause protein mislocalization. Strategies to disrupt XPO1 activity fully restore ETV6 P214L protein nuclear localization and transcription regulation activity. Mechanistic insight inspired the design of a humanized ETV6 mice, which we employ to demonstrate that the germline P214L mutation is sufficient to elicit severe defects in thrombopoiesis and hematopoietic stem cell maintenance. Beyond ETV6, we employed computational methods to uncover rare disease-associated missense mutations in unrelated proteins that create a nuclear export signal to disrupt protein function. Thus, missense mutations that operate through this mechanism should be predictable and may suggest rational therapeutic strategies for associated diseases.

biochemistry↗