RNA sculpting by the primordial Helix-clasp-Helix–Strand-Loop (HcH–SL) motif enforces chemical recognition enabling diverse KH domain functions

In all domains of life, the ancient K homology (KH) domain superfamily is central to RNA processes including splicing, transcription, posttranscriptional gene regulation, signaling, and translation. Proteins with 1 to 15 KH domains bind single-strand (ss) RNA or DNA with base sequence specificity. Here, we examine over 40 KH domain experimental structures in complex with nucleic acid (NA) and define a novel Helix-clasp-Helix–Strand-Loop (HcH–SL) NA recognition motif binding 4 to 5 nucleotides using 10 to 18 residues. HcH–SL includes and extends the Gly-X-X-Gly (GXXG) signature sequence “clasp” that brings together two helices as an ∼90° helical corner. The first helix primarily provides side chain interactions to unstack and sculpt 2 to 3 bases on the 5′ end for recognition of sequence and chemistry. The clasp and second helix amino dipole recognize a central phosphodiester. Following the helical corner, a beta strand and its loop extension recognize the two 3′ nucleotides, primarily through main chain interactions. The HcH–SL structural motif forms a right-handed triangle and concave functional interface for NA interaction that unexpectedly splays four bound nucleotides into conformations matching RNA recognition motif (RRM) bound RNA structures. Evolutionary analyses and its ability to recognize base sequence and chemistry make HcH–SL a primordial NA binding motif distinguished by its binding mode from other NA structural recognition motifs: helix-turn-helix, helix-hairpin-helix, and beta strand RRM motifs. Combined results explain its vulnerability as a viral hijacking target and how mutations and expression defects lead to diverse diseases spanning cancer, cardiovascular, fragile X syndrome, neurodevelopmental disorders, and paraneoplastic disease.