Biology subjects

Gordon, B. H.

Publications and source records attributed to Gordon, B. H..

3 recordsLinked to original sources

bioRxiv2025

Non-Canonical RNA Binding of Human La-related Protein 6

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 5SL by LARP6 that is related to collagen biosynthesis in fibrosis and associated pathologies.

biophysics↗

bioRxiv2024

Deciphering the Binding of 5' Stem Loop RNA to the La Domain of Human LARP6

Human La-related protein 6 (HsLARP6) regulates the highly organized biosynthesis of type I procollagen polypeptides and affects proper assembly of procollagen peptides into heterotrimers of type I procollagen. HsLARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5 stem loop (5SL) motif found in type I and III collagen messenger RNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic propensity of the La domain of HsLARP6 to aggregate hampers studies probing the molecular basis of biologically- and disease-relevant structure-function relationship, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effect of the protein construct length and RNA binding on protein stability. C-terminal truncations greatly impact protein stability, while N-terminal truncations have little to none effect on protein aggregation and RNA binding. When in complex with its cognate 5SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50x longer than the unbound state, under identical conditions. These results provide a significant platform for further studies of the molecular recognition of 5SL by HsLARP6.

molecular biology↗

bioRxiv2020

Characterization of sequence specific binding of LARP6 to the 5' stem-loop of type I collagen mRNAs and implications for rational design of antifibrotic drugs

Excessive synthesis of type I collagen characterizes fibrotic diseases. Binding of LARP6 to the 5 stem-loop (5SL) of collagen mRNAs regulates their translation and the high rate of biosynthesis in fibrosis. LARP6 needs two domains to form stable complex with 5SL RNA, the La-domain and the juxtaposed RRM domain (jointly called the La-module). We describe that the La-domain of LARP6 is necessary and sufficient for recognition of 5SL in sequence specific manner. The three amino acid motif, RNK, located in the flexible loop which connects the second -helix to the {beta}-sheet of the La domain is critical for binding. Mutation of any of these three amino acids abolishes the binding of La-domain to 5SL. The major site of crosslinking of LARP6 to 5SL RNA was mapped to this motif. The RNK motif is not found in other LARPs, which can not bind 5SL. Presence of RRM increases the stability of complex between La-domain and 5SL RNA and RRM domain does not make extensive contacts with 5SL RNA. We propose a model in which the initial recognition of 5SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.

molecular biology↗