Gordon, B. H.; Ogunkunle, V. S.; Silvers, R.

La-related proteins (LARPs) are RNA-binding proteins that are involved in a variety of disease-related processes. Most LARPs recognize short single-stranded poly(U/A) motifs via a conserved hydrophobic pocket. Human LARP6 (HsLARP6) is an exception, binding a structured 5\' stem-loop (5\'SL) that controls type I collagen translation and fibroproliferative disease progression. Here, we present the de novo solution NMR structure of the La domain of HsLARP6 in the bound state. Chemical shift perturbation, solvent paramagnetic relaxation enhancement, intermolecular NOEs and targeted mutagenesis converge on a previously unknown binding interface that integrates electrostatic and hydrophobic contacts with shape complementarity in 5\'SL binding. This non-canonical interface enables the La domain to discriminate 5\'SL RNA from homopolymeric or purely helical hairpin RNAs with low-nanomolar affinity, overturning earlier views that the adjacent RRM is required for recognition. The structure provides the first molecular model for 5\'SL recognition and expands the paradigm of La-mediated RNA binding beyond 3\'-terminal oligo-U/A motifs. These insights provide the biophysical framework for molecular recognition of 5\'SL by LARP6 that is related to collagen biosynthesis in fibrosis and associated pathologies.