Biology subjects

Schweimer, K.

Publications and source records attributed to Schweimer, K..

8 recordsLinked to original sources

bioRxiv2026

Transient interdomain interactions shape the conformational ensemble governing RNA recognition by the tandem RRMs of Sex-lethal

RNA recognition motif (RRM) proteins frequently contain multiple RNA-binding domains connected by flexible linkers, yet the contribution of transient interdomain interactions to RNA recognition remains incompletely understood. Here, we investigated the structural organization of the tandem RRMs of the Drosophila melanogaster splicing regulator Sex-lethal (Sxl) using solution NMR spectroscopy in combination with rational protein engineering, restrained docking and RNA-binding studies. Progressive extension of the native interdomain linker resulted in a gradual decrease in rotational coupling between the two RRMs and continuous chemical shift changes, demonstrating that the RNA-free protein samples a dynamic conformational ensemble rather than behaving as two independently tumbling domains. NMR-guided docking identified a compact arrangement compatible with the experimental data and suggested a transient interface partially overlapping the RNA-binding surfaces. Surprisingly, a mutant designed to weaken this interface produced the opposite effect: instead of increasing interdomain mobility, it exhibited enhanced rotational coupling while remaining natively folded, indicating a redistribution of the conformational ensemble rather than disruption of the domain architecture. Both linker extension and the mutant reduced RNA-binding affinity, and the mutant additionally diminished sequence discrimination, demonstrating that perturbations shifting the conformational equilibrium in either direction compromise RNA recognition. Together, our results demonstrate that RNA recognition by Sxl is governed not by a single apo structure but by a finely balanced conformational ensemble, and that perturbing this equilibrium in either direction compromises high-affinity and sequence-selective RNA binding.

biophysics↗

bioRxiv2026

Multimodal phenotyping defines variant-to-function maps for RBM20 in dilated cardiomyopathy

Multiplex assays of variant effects have linked thousands of genotypes to fitness effects, yet we lack profound understanding of how variants impact molecular phenotypes. Here, we introduce a deep mutational scanning framework that quantifies disease-determining molecular phenotypes in human cells, allowing readouts of protein localization and splicing regulatory function at scale. Applied to the dilated cardiomyopathy (DCM)-associated protein RBM20, we profiled [~]4,300 amino acid substitutions across disease-linked protein domains. Complemented by structure-function investigations of RBM20 bound to its nuclear import receptor TNPO3, we discover new variant hotspots affecting protein function. Finally, we systematically probed nuclear relocalization to identify variants that may be amenable to this therapeutic strategy. Together, we create comprehensive variant-to-function maps that predict variant impact, enhance clinical interpretation, and stratify RBM20-mediated DCM into mechanistically distinct therapeutic classes.

genomics↗

bioRxiv2026

dsRBD Redesign: A Targeted Strategy for Inhibition of RNA Helicase DHX9

The RNA helicase DHX9 is essential for genomic stability, transcription, translation regulation and other RNA-related processes. DHX9 has emerged as a therapeutic target for cancer treatment as its expression levels are elevated in several different cancer types. Moreover, tumor cells exhibit a strong dependence on DHX9, making its inhibition an effective strategy for tumor regression. As RNA helicases are conserved enzymes, unique features need to be targeted to minimize side effects. Here, we identified an autoregulatory interface between the DHX9 helicase core and double-stranded RNA binding domain 2 (dsRBD2) ideal for a highly specific inhibition of DHX9. By targeting the proposed dsRBD2-core interface in DHX9, we aim to specifically inhibit DHX9 helicase activity by preventing dsRBD2 binding to the helicase core. We developed a protein binder design-based strategy targeting the dsRBD2-core interface of DHX9 by computationally designing novel dsRBDs. By binding this interface without engaging RNA, the dsRBD designs prevent dsRBD2-core interaction and inhibit DHX9 helicase activity. Our strategy of redesigning autoregulatory protein domains as inhibitors offers a computationally efficient alternative to larger-scale library generation and provides a flexible framework applicable to a wide range of therapeutic targets.

biochemistry↗

bioRxiv2026

Electrostatic buffering of non-native interactions in the transition state ensemble for binding of intrinsically disordered proteins

Intrinsically disordered protein regions (IDRs) establish highly specific protein-protein interactions that play key roles in cell signaling. Many IDRs adopt an ordered structure upon binding to folded protein domains, but the energy landscape for coupled folding and binding (CFB) is poorly understood. Here, we elucidate the energy landscape for CFB using the LxCxE motif from the human papillomavirus E7 protein (LxCxEWT) as a minimal model system. Kinetic and structural analysis uncovers strong compensatory energetics whereby stabilizing electrostatic interactions counterbalance energetic frustration in the rate-limiting step for CFB. This mechanism, which we refer to as electrostatic compensation, enables a dynamic search for native contacts while preventing complex dissociation. A global analysis reveals energetic frustration in earliest steps of CFB for many IDRs. Electrostatic compensation may be a widespread mechanism evolved to allow fine-tuning of the affinity and specificity of IDR interactions, which dictates functional selection for charge content within IDRs.

biophysics↗

bioRxiv2024

The Drosophila RNA binding protein Hrp48 binds a specificRNA sequence of the msl-2 mRNA 3' UTR to regulatetranslation

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3 untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3 UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

biophysics↗

bioRxiv2023

Hydrophobic Clusters Direct Folding of a Synthetic Chimeric Protein

Extant proteins frequently share sub-domain sized fragments, suggesting that among other mechanisms, proteins evolved new structure and functions via recombination of existing fragments. While the role of protein fragments as evolutionary units is well-established, their biophysical features necessary for generating a well-folded and stable protein are not clearly understood. In order to probe how fragments determine foldability and stability of recombined proteins, we investigated the stability, folding and dynamics of a synthetic chimera created by fusion of fragments of the chemotactic response regulator protein CheY that belongs to the flavodoxin-like fold and imidazole glycerol phosphate synthase from histidine biosynthesis (HisF) which harbors the TIM-barrel fold. The chimera unfolds via an equilibrium intermediate. Mutation of a glycine residue present at the interface of the CheY and HisF fragments to a valine abrogates the equilibrium intermediate while mutation to isoleucine dramatically increases the native state kinetic stability without any significant change in the folding rate. Parts of the fragment interface in the chimera are found to be conformationally dynamic while hydrophobic mutations globally increase its conformational rigidity. We hypothesize that the hydrophobic mutation improves sidechain packing in a large cluster of isoleucine, leucine and valine (ILV) residues that spans the fragment interface. We also extrapolate that inheritance of large ILV clusters from parent proteins could be a key determinant of successful fragment recombination.

biophysics↗

bioRxiv2022

Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins

The two-domain protein RfaH, a paralog of the universally conserved NusG/Spt5 transcription factors, is regulated by autoinhibition coupled to the reversible conformational switch of its 60-residue C-terminal KOW domain between an -hairpin and a {beta}-barrel. In contrast, NusG/Spt5-KOW domains only occur in the {beta}-barrel state. To understand the principles underlying the drastic fold switch in RfaH, we elucidated the thermodynamic stability and the structural dynamics of two RfaH- and four NusG/Spt5-KOW domains by combining biophysical and structural biology methods. We find that the RfaH-KOW {beta}-barrel is thermodynamically less stable than that of most NusG/Spt5-KOWs and we show that it is in equilibrium with a globally unfolded species, which, strikingly, contains two helical regions that prime the transition towards the -hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.

biophysics↗

bioRxiv2018

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH: ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

biochemistry↗