Biology subjects

Schwan, M.

Publications and source records attributed to Schwan, M..

3 recordsLinked to original sources

bioRxiv2025

DNA-encoded Library Screening Uncovers Potent DNMT2 Inhibitors Targeting a Cryptic Allosteric Binding Site

The human RNA methyltransferase DNMT2 is thought to be involved in various pathophysiological processes, yet, a major challenge in drug targeting DNMT2 is given by the fact that current SAH-derived inhibitors have poor target selectivity and limited cellular permeability. In this study, we have performed a DNA-encoded library (DEL) screening on DNMT2 yielding five non-SAH-like hit structures, three of which feature a peptidomimetic scaffold. All DEL hits could be validated by orthogonal biophysical and biochemical assays for DNMT2 binding. At the same time, the lead structure did not interact with related methyltransferases from the DNMT and NSUN families highlighting an unmatched DNMT2-targeting selectivity profile. Subsequent crystallographic studies revealed the unique ligand binding mode including an active site loop rearrangement and the formation of a cryptic allosteric binding pocket able to modulate the enzymatic activity by non-covalent DNMT2 dimerization. Based on the crystallographic results, we performed a structure-activity relationship study around the inhibitor lead structure resulting in an optimized DNMT2 inhibitor (KD=3.04 {micro}M), which was able to reduce m5C levels in MOLM-13 tRNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/632061v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1bf92b3org.highwire.dtl.DTLVardef@38498borg.highwire.dtl.DTLVardef@1645740org.highwire.dtl.DTLVardef@9c61d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Structures of aberrant spliceosome intermediates on their way to disassembly

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans1. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses2,3, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-EM structures of post-Bact spliceosome intermediates from S. pombe primed for disassembly. We identify the DEAH-box helicase - G patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an over-stabilization of the U5 loop I interaction with the 5 exon leading to a single nucleotide insertion at the 5splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.

biochemistry↗

bioRxiv2022

Constitutive production of flagellar proteins is required for proper flagellation in Shewanella putrefaciens

Flagella are multiprotein complexes whose assembly and positioning requires complex spatiotemporal control. Flagellar assembly is thought to be controlled by several transcriptional tiers, which is mediated through various master regulators. Here, we revisited the regulation of flagellar genes in polarly flagellated gammaproteobacteria by the regulators FlrA, RpoN ({sigma}54) and FliA ({sigma}28) in Shewanella putrefaciens CN-32 at the transcript and protein level. As expected, strict control at both levels occurred for for highly abundant flagellar proteins, including the building blocks for the outer rings, rod, hook and filament. In contrast, a number of regulatory and structural proteins were always present also in the absence of the main regulators. Initiation of flagella assembly and motor activation likely relies on the abundance control of only few structural key components required for formation of the MS- and C-ring and the flagellar type III secrection system. We identified {sigma}70-dependent promoters driving constitutive expression of some flagellar genes including the regulators of flagellar number and positioning, FlhF and FlhG. Reduction of the constitutive expression levels resulted in emergence of hyperflagellation. Thus, basal expression and presence of flagellar proteins is required for proper flagellation, which adds a deeper layer to the regulation of flagellar synthesis and assembly. SignificanceThe tier-based transcriptional regulation underlying bacterial flagella synthesis is - with certain variations - well-established in various species. Here we show that initiation and proceeding of flagellar synthesis can be simply based on the control of some key components and highly abundant building blocks. We further identified a 'tier zero, a set of constitutively produced flagellar regulators and building blocks, which is required, for example, to maintain the flagellar counter. We expect this not only to apply to our model species Shewanella, but also to other flagella regulation systems in bacteria.

microbiology↗