The divergent LARP1 PAM2 motif adopts a non-canonical conformation for MLLE binding

Cytoplasmic poly(A)-binding protein (PABPC1) binds the mRNA poly(A) tail via its RNA recognition motifs (RRMs) and recruits factors involved in mRNA metabolism through its C-terminal MLLE domain. The MLLE domain recognizes short PAM2 motifs found in a wide range of regulatory proteins known as PAM2-containing proteins (PACs). Members of the La-related protein (LARP) family, specifically LARP1 and LARP4, which are involved in poly(A) tail protection, contain atypical PAM2 sequences that diverge from the canonical consensus yet retain MLLE binding. The molecular basis of this interaction is not fully understood for LARP1. Here, we combined nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC), and AlphaFold3 modeling to characterize the interaction between the LARP1 PAM2 motif and the MLLE domain of PABPC1. NMR chemical shift perturbations and heteronuclear NOE analyses defined the MLLE-binding segment, i.e. PAM2 motif, within LARP1. Mutagenesis studies and ITC confirmed the essential role of phenylalanine 496 for MLLE binding. AlphaFold3 modeling corroborated these findings with the prediction of a single turn α-helix in the PAM2 motif. Together, these results define a non-canonical PAM2-MLLE interaction and reveal unexpected plasticity in the recognition of PAM2 motifs by the MLLE domain.