Biology subjects

Sprangers, R.

Publications and source records attributed to Sprangers, R..

3 recordsLinked to original sources

bioRxiv2024

Conformational modulation of a mobile loop controls catalysis in the (βα)8-barrel enzyme of histidine biosynthesis HisF

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site {beta}11-loop (loop1) of the ({beta})8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N-[(5-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of ({beta})8-barrel enzymes and beyond. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/600150v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@9c5db4org.highwire.dtl.DTLVardef@1cc090dorg.highwire.dtl.DTLVardef@6646d7org.highwire.dtl.DTLVardef@b4e1f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Beyond static structures: quantitative dynamics in the eukaryotic RNA exosome complex

Molecular machines play pivotal roles in all biological processes. Most structural methods, however, are unable to directly probe molecular motions. Here, we demonstrate that dedicated NMR experiments can provide quantitative insights into functionally important dynamic regions in very large asymmetric protein complexes. We establish this for the 410 kDa eukaryotic RNA exosome complex that contains ten distinct protein chains. Methyl-group and fluorine NMR experiments reveal site-specific interactions among subunits and with an RNA substrate. Furthermore, we extract quantitative insights into conformational changes within the complex in response to substrate and subunit binding for regions that are invisible in static cryo-EM and crystal structures. In particular, we identified a flexible plug region that can block an aberrant route of RNA towards the active site. Based on molecular dynamics simulations and NMR data we provide a model that shows how the flexible plug is structured in the open and closed conformations. Our work thus demonstrates that a combination of state-of-the-art structural biology methods can provide quantitative insights into large molecular machines that go significantly beyond the well-resolved and static images of biomolecular complexes, thereby adding the time domain into structural biology.

molecular biology↗

bioRxiv2022

A conserved and tunable mechanism for the temperature-controlled condensation of the translation factor Ded1p

Heat shock promotes the assembly of translation factors into condensates to facilitate the production of stress-protective proteins. How translation factors detect heat and assemble into condensates is not well understood. Here, we investigate heat-induced condensate assembly by the translation factor Ded1p from five different fungi, including Ded1p from Saccharomyces cerevisiae. Using targeted mutagenesis and in vitro reconstitution biochemistry, we find that heat-induced Ded1p assembly is driven by a conformational rearrangement of the folded helicase domain. This rearrangement determines the assembly temperature and the assembly of Ded1p into nanometer-sized particles, while the flanking intrinsically disordered regions engage in intermolecular interactions to promote assembly into micron-sized condensates. Using protein engineering, we identify six amino acid substitutions that determine most of the thermostability of a thermophilic Ded1p ortholog, thereby providing a molecular understanding underlying the adaptation of the Ded1p assembly temperature to the specific growth temperature of the species. We conclude that heat-induced assembly of Ded1p into translation factor condensates is regulated by a complex interplay of the structured domain and intrinsically disordered regions which is subject to evolutionary tuning.

biochemistry↗