Matsingos, C. | bioRxiv Science

bioRxiv2026

Molecular basis of Salla Disease: R39C Mutation Effects on the Lysosomal Transporter Sialin

Salla disease is caused by a genetic mutation in sialin, a lysosomal membrane transporter, which exports sialic acid from lysosomes. Substrate translocation occurs via a rocker-switch mechanism that alternately exposes the substrate-binding site to the lysosomal lumen and the cytosol. The pathogenic mutation R39C found in most Salla disease patients decreases the lysosomal localisation and the transport activity. In this study, we used computational and mutagenesis approaches to elucidate the molecular effects of the R39C mutation. Using three-dimensional models of human sialin in the lumen-open (LO) and cytosol-open (CO) states combined with the mutagenesis of selected residues, we identify a critical "triplet" motif comprising R39, E194, and E262, which is associated with an ionic lock formed between K197 and D350 in the LO conformation. Molecular dynamics simulations suggest that the electrostatic triplet negatively modulates the ionic lock, and are consistent with a strengthened ionic lock in R39C sialin, potentially favouring the LO state. To assess the global effects of the R39C mutation, we computed dynamic cross-correlation matrices and identified correlation patterns consistent with an allosteric coupling between the ionic lock K197/D350 and the region surrounding the sialic acid binding site in wild-type sialin, whereas in the LO state of R39C sialin, this communication preferentially bypasses this region. Therefore, the R39C mutation may impede the LO to CO conformational transition required for sialic acid transport, providing a plausible mechanistic framework for the decreased transport activity, and possibly the decreased lysosomal localisation, observed in Salla disease. HighlightsO_LIThe R39 residue participates in an interaction triplet, which negatively regulates an ionic lock stabilising the lumen-open conformation C_LIO_LIThe R39C mutation is associated with a stronger ionic lock in the simulations, and may favour the lumen-open state C_LIO_LICorrelation network analysis suggests an allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LIO_LIThe R39C mutation alters the inferred allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/719580v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1bf7144org.highwire.dtl.DTLVardef@1a53ab8org.highwire.dtl.DTLVardef@b2249forg.highwire.dtl.DTLVardef@1827244_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

TrIPP: a Trajectory Iterative pKa Predictor

The protonation propensity of ionisable residues in proteins can change in response to changes in the local residue environment. The link between protein dynamics and pKa is particularly important in pH regulation of protein structure and function. Here, we introduce TrIPP (Trajectory Iterative pKa Predictor), a Python tool to monitor and analyse changes in the pKa of ionisable residues during Molecular Dynamics simulations of proteins. We show how TrIPP can be used to identify residues with physiologically relevant variations in their predicted pKa values during the simulations, and link them to changes in the local and global environment. TrIPP is available at https://github.com/fornililab/TrIPP.

bioinformatics↗

bioRxiv2023

Elucidating the activation mechanism of the proton-sensing GPR68 receptor

GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) involved in a variety of physiological processes and disorders including neoplastic pathologies. While GPR68 and few other GPCRs have been shown to be activated by a decrease in the extracellular pH, the molecular mechanism of their activation remains largely unknown. In this work, we used a combined computational and in vitro approach to provide new insight into the activation mechanism of the receptor. Molecular Dynamics simulations of GPR68 were used to model the changes in residue interactions and motions triggered by pH. Global and local rearrangements consistent with partial activation were observed upon protonation of the inactive state. Selected extracellular histidine and transmembrane acidic residues were found to have significantly upshifted pKa values during the simulations, consistently with their previously hypothesised role in activation through changes in protonation state. Moreover, a novel pairing between histidine and acidic residues in the extracellular region was highlighted by both sequence analyses and simulation data and tested through site-directed mutagenesis. At last, we identified a previously unknown hydrophobic lock in the extracellular region that might stabilise the inactive conformation and regulate the transition to the active state.g

molecular biology↗