Alternative PAM2 Motif Architecture in La-related Protein 1

Blaine H. Gordon^1,2, Robert Silvers^1,2

1. Department of Chemistry and Biochemistry, Florida State University
2. Institute of Molecular Biology, Florida State University

La related proteins (LARPs) are a superfamily of RNA binding protein which associate with a variety of RNA substrates using their La module, a highly conserved La domain in tandem with a downstream ribonucleotide recognition motif (RRM). La-related protein 1A and 1B are the only LARP superfamily members found to lack a RRM entirely, as determined by AlphaFold predictions and NMR studies performed in our lab and in others,. LARP1A/B function to recognize both the 5’-Gppp cap of terminal oligopyrimidine (TOP)-containing mRNA using its novel DM15 domain, as well as their 3’ poly(A) tails using its La motif, in an mTOR kinase-dependent manner.

The C-terminal (MLLE) domain of PABP, an a-helical domain named for the highly conserved KITGMLLE motif, serves to recruit components of mRNA stabilization or turnover by recognizing PAM2 motifs found in unstructured regions of relevant regulatory protein. PAM2 motifs share a 12 residue consensus sequence of the form xx^L/_PNxxAxEFxP, with the 3^rd position hydrophobic residue and 10^thposition phenylalanine each being required for high affinity binding. The LARP1 PAM2 sequence (a.a. 496-508), FSQLLNCPEFVPR, utilizes an alternative sequence organization to binding MLLE using the binding site recognized by archetypal PAM2 motifs from polyadenylate interaction proteins 1 and 2 (PAIP1 and PAIP2, resp.).

Despite previous work characterizing the importance of the 1^st position phenylalanine in the LARP1 PAM2 motif for high affinity binding to MLLE, the binding mode of this residue and otheralternative positions for this atypical PAM2 motif remain unclear. To address this gap in knowledge, we herein provide quantitative and qualitative dissections of LARP1 PAM2 motif interaction with the MLLE through NMR spectroscopy and molecular dynamics simulations in comparison with archetypal PAM2 motifs from PABP-interaction proteins 1 and 2 (PAIP1 and PAIP2, resp.). Through NMR titrations of PAM2 peptides against [¹⁵N]-Labeled MLLE(544-626) and alchemical molecular dynamics simulations, we conclude that the LARP1 PAM2 uses its initial F496 to recapitulate the function of the 3^rd position hydrophobic residue of genuine PAM2 motifs. Moreover, the isolated 12 amino acid PAM2 motifs of PAIP1 and PAIP2 each bind the MLLE domain with a low micromolar affinity, only slightly weaker than an the full length protein. The isolated PAM2 motif of LARP1 binds MLLE with a low millimolar affinity which facilitated a detailed analysis of the peptide:protein interaction by NMR. Extending the C-terminal sequence of the LARP1 PAM2 motif peptide reduced the Kd by a factor over 1000-fold, then achieving a the low micromolar affinity to MLLE seen in nearly all PAM2-containing protein. From these result we assert LARP1 as an example of a PAM2-like sequence defined by principle interactions in three segments which each occupy individual hydrophobic binding sites across the 5-helix landscape of the PABP C-terminal domain.