Biology subjects

Puyo-Fourtine, J.

Publications and source records attributed to Puyo-Fourtine, J..

2 recordsLinked to original sources

bioRxiv2026

Origins of reactivity in SAM-utilizing ribozyme SAMURI-catalyzed RNA alkylation

Unlocking the design principles of programmable RNA catalysts capable of sitespecific chemical modification is critical for expanding the functional and therapeutic potential of RNA. The SAM analogue-utilizing ribozyme (SAMURI) enables sitespecific RNA alkylation using either S-adenosylmethionine (SAM) or the synthetic cofactor propargylic Se-2,6-diaminopurinribosyl-selenomethionineamide (ProSeDMA), yet the molecular determinants of its reactivity remain incompletely understood. Here, we combined molecular dynamics, 3D-RISM solvation analysis, alchemical free-energy calculations, quantum pKa shift predictions, and ab initio QM/MM free-energy simulations to characterize the conformational and electronic factors that govern catalysis. Simulations show that, although the global fold of SAMURI remains stable in solution, the formation of catalytically competent near-attack configurations is rare, indicating that the observed rate depends on access to a minor fraction of these reactive conformations (freact). A putative Mg2+ binding site between the SAM carboxylate and the G30 phosphate, together with a hydrogen bond between the cofactor -amine and U8:O2, enriches freact. QM/MM simulations support an SN2-like alkyl transfer mechanism and show that ProSeDMA reacts more readily than SAM primarily due to its more favorable electronic leaving-group properties that enhance the intrinsic rate (kint). Atomic substitutions at A52 that tune the N3 pKa enhance nucleophilicity, further lower the activation barrier, and increase kint. Together, these results show that SAMURI catalysis is governed by a combination of conformational preorganization and electronic effects, providing a framework to guide the design of new programmable RNA alkyltransferases. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/720726v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@90e0c1org.highwire.dtl.DTLVardef@193b8d6org.highwire.dtl.DTLVardef@14c0d49org.highwire.dtl.DTLVardef@1fc6793_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Sticky salts: overbinding of monovalent cations to phosphorylations in all-atom forcefields

Phosphorylation is a major post-translational modification, which is involved in the regulation of the dynamics and function of Intrinsically Disordered Proteins (IDPs). We recently characterized a phenomenon, which we termed n-Phosphate collaborations (nP-collabs), where bulk cations form stable bridges between several phosphoresidues in all-atom molecular dynamic simulations. nP-collabs were found to be sensitive to the combination of forcefields and cation types. Here, we attempt to assess the physical relevance of these nP-collabs by evaluating the strength of the cation/phosphate interaction through osmotic coefficient ({phi}) calculations on the model [Formula] and [Formula] salts, using different classical forcefields for phosphorylations. All force-fields were found to overestimate the strength of the interaction to various degrees. We thus designed new parameters for CHARMM36m and AmberFF99SB-ILDN using the Electronic Continuum Correction (ECC) approach, which provide remarkable agreement for{phi} values for both cation types and over a range of concentrations. We provide a preliminary test of these ECC parameters for phosphorylations by simulating the sevenfold-phosphorylated rhodopsin peptide 7PP and comparing secondary chemical shifts to experimental data. Conformational ensembles resulting from the ECC-derived phosphorylated forcefields display both qualitative and quantitative improvements with regard to full-charge forcefields. We thus conclude that long-lasting nP-collabs are artifacts for classical forcefields born from the lack of explicit polarization, and propose a possible computational strategy for the extensive parameterization of phosphorylations. The presence of long-lived nP-collabs in simulations produced using classical forcefields is therefore a serious concern for the accurate modelling of multiphosphorylated peptides and IDPs, which are at the center of research questions regarding neurodegenerative diseases such as Alzheimers or Parkinsons.

biochemistry↗