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.

TRIM2 is a mammalian E3 ligase with particularly high expression in Purkinje neurons, where it contributes to neuronal development and homeostasis. The understanding of ubiquitin E3 ligase function hinges on thoroughly identifying their cellular targets, but the transient nature of signaling complexes leading to ubiquitination poses a significant challenge for detailed mechanistic studies. Here, we tailored a recently developed ubiquitin-specific proximity labeling tool to identify substrates of TRIM2 in cells. We show that TRIM2 targets proteins involved in the endolysosomal pathway. Specifically, we demonstrate using biochemical and structural studies, that TRIM2 ubiquitinates TMEM106B at lysine residues located in the cytosolic N-terminal region. Substrate recognition involves a direct interaction between TRIM2 and a newly identified zinc-coordination motif in TMEM106B that mediates homodimerization, is required for specific protein-protein interactions, and lysosomal size regulation. We found that in addition to catalysis, the tripartite motif is involved in substrate recruitment. Our study thus contributes a catalog of TRIM2 effectors and identifies a previously unrecognized regulatory region of TMEM106B crucial to its function.

Heteroleptic coordination cages enable not only a high control over the cavity properties but also multifunctionality. However, their synthesis is far from trivial and often relies on precious 4d and 5d transition metals such as Pd(II). The transfer to more cost-effective, earth abundant metal ions such as Zn(II) is highly desirable for increasing their application potential in real-life scenarios. In this work, we report on a self-assembly strategy employing the ditopic Zn(II) bis-salphen unit Zn2L in combination with tritopic pyridyl-based ligands to construct novel prismatic cages. As a proof of concept, two distinct cages are presented, utilizing either a phenyl- or triazine-based tritopic ligand (cage 1 and cage 2, respectively). Both cages exhibit remarkable stability at low concentrations while displaying dynamic behaviour at high concentrations. Investigation of their photophysical properties reveals a striking "on-off" emission behaviour: the phenyl-based cage 1 features a significant orange emission (Φ = 6%), while cage 2 is non-emissive. This emission quenching can be attributed to the electron-withdrawing nature of the triazine ligand, which dominates the electronic relaxation pathway of the Zn2L unit. Although both cages successfully bind aromatic guests in host-guest studies, their encapsulation results in partial cage decomposition, which prevents further investigation of whether cage 1 qualifies as an optical sensor material. However, the findings herein introduce a versatile route to emissive bis-salphen coordination cages, marking a significant step towards developing new heteroleptic zinc(II) cages for optical sensing.

Intrinsically disordered protein regions (IDRs) mediate key steps in cellular signaling but the nature of the energy barriers crossed by IDRs upon association and the energetic features of the transition state ensemble (TSE) for binding remain elusive. Short linear motifs (SLiMs) are small functional units found within IDRs that fold upon binding to globular domains. Here, we use the LxCxE SLiM from the human papillomavirus E7 protein (LxCxEWT) binding to the retinoblastoma (Rb) protein as a minimal model system for an IDR-domain interaction. We combine extensive mutagenesis with electrostatic dissection to gain information on the binding TSE energetics and rule out ground state effects using Far-UV CD and NMR. This approach uncovers strong compensatory energetics whereby stabilizing electrostatic interactions play a key role buffering multiple weakly destabilizing non-native interactions in the binding TSE for LxCxEWT. Electrostatic buffering may enable a dynamic search for native contacts and fine tuning of the TSE energetics. A global analysis of 177 mutations reveals non-native energetics in the binding TSE of many IDRs, suggesting that electrostatic buffering may be a widespread phenomenon that dictates functional selection for charge content within IDRs. ### Competing Interest Statement The authors have declared no competing interest. Agencia Nacional de Promoción Científica y Tecnológica, 2019-02119, 2021-001027 European Union Horizon Europe MSCA Staff Exchange, 778247

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.

The global production of hazelnuts is rising, but hazelnut allergies remain common and severe. The PR-10-like allergen Cor a 1, found in hazel pollen and nuts, consists of different isoallergens with over 67 % sequence identity. Cor a 1 binds plant secondary metabolites like the diglycosylated flavonoid Q3O-(Glc)-Gal. Understanding the ligand binding of Cor a 1 isoallergens is crucial to understand their physiological and allergenic properties. We identified physiological mono- and diglycosylated flavonoids from hazel pollen by UHPLC/QTOF-MS and NMR spectroscopy and investigated their binding to Cor a 1 isoallergens by NMR HSQC experiments and ITC titrations. Screening analyses revealed different binding properties of isoallergens. Whereas the isoallergen Cor a 1.0501 bound only monoglycosylated flavonoids, Cor a 1.0401 also bound diglycosylated flavonoids. Protein/ligand complex structures based on NMR NOESY and computational docking experiments revealed different orientations of mono- vs. diglycosylated flavonoids in the amphiphilic pocket and specific binding of disaccharide derivatives.

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 .

De novo protein design is of fundamental interest to synthetic biology, with a plethora of computational methods of various degrees of generality developed in recent years. Here, we introduce AlphaDesign, a hallucination-based computational framework for de novo protein design developed with maximum generality and usability in mind, which combines AlphaFold with autoregressive diffusion models to enable rapid generation and computational validation of proteins with controllable interactions, conformations and oligomeric state without the requirement for class-dependent model re-training or fine-tuning. We apply our framework to design and systematically validate in vivo active inhibitors of a family of bacterial phage defense systems with toxic effectors called retrons, paving the way towards efficient, rational design of novel proteins as biologics.

The understanding of ubiquitin E3 ligase function hinges on thoroughly identifying their cellular targets, but the transient nature of signaling complexes leading to ubiquitination poses a significant challenge for detailed mechanistic studies. TRIM2 and TRIM3 are paralogous mammalian E3 ligases with particularly high expression in the brain, where they contribute to neuronal development and homeostasis. Here, we tailored recently developed ubiquitin-specific proximity labelling tools to identify substrates of TRIM2 and TRIM3 activity. We show that despite their high amino acid sequence identity, the ligases have distinct intracellular dynamics, binding partners, and ubiquitination substrates. Using biochemical and structural studies, we show that TRIM2 ubiquitinates the lysosomal protein TMEM106B at lysine residues located in the cytosolic N-terminal region. Substrate recognition involves a direct interaction between TRIM2 and a newly identified zinc-coordination motif in TMEM106B that mediates homodimerization and is required for lysosomal size regulation. We found that in addition to catalysis, the tripartite motif is involved in substrate recruitment, and we provide insights into the assembly of the ubiquitination complex. Deletion of TRIM2/TRIM3 in mouse embryonic stem-cell derived neurons impacted the extracellular matrix composition, likely through acting on the endolysosomal pathway. Our study thus contributes a catalogue of TRIM2 and TRIM3-associated effectors and supports a key role at the interface of vesicle trafficking and the cytoskeleton.

AbstractRepression ofmsl-2mRNA translation is essential for viability ofDrosophila melanogasterfemales 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 themsl-2transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction withmsl-2are 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 ofmsl-23’ 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 tomsl-2.

Surface-compartmentalized polymer micelles (Janus and patchy micelles) have gained increasing attention as their unique properties open the way for various applications. While Janus micelles have been extensively studied, e.g. as compatibilizers in polymer blends, there are hardly any reports on the use of patchy micelles. In this study, we show that spherical micelles with a polylactide stereocomplex (SC) core and a patch-like microphase separated polystyrene/poly(tert-butyl methacrylate) (PS/PtBMA) corona are efficient compatibilizers for highly immiscible PS/PtBMA blends. The patchy SC micelles, prepared by stereocomplex-driven self-assembly (SCDSA) of enantiomeric diblock copolymers, improved the homogeneity of the blends and led to a significant reduction of the PS droplet size. We further employed SCDSA to selectively incorporate a fluorescent dye inside the SC micelle core without changing the shape or chemistry of the patchy corona. This allows the use of confocal scanning fluorescence microscopy to localize the patchy SC micelles, being predominantly assembled at the PS/PtBMA blend interface. Interestingly, the reduction in PS droplet size was comparable for blends compatibilized with patchy SC micelles and Janus micelles, but only for patchy SC micelles a monomodal droplet size distribution could be achieved. The outstanding interfacial activity of the patchy SC micelles can be attributed to their adaptive corona structure, resulting in a selective swelling/collapse of the respective miscible/immiscible corona patches at the blend interface. Patchy spherical micelles prepared via stereocomplex-driven self-assembly are applied as efficient compatibilizers for highly immiscible polystyrene/poly(tert-butyl methacrylate) blends.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in metabolic regulation and aging-related dysfunctions. The nuclear isoform Sirt1 deacetylates histones and transcription factors and contributes, e.g., to brain and immune cell functions. Upon infection by human immunodeficiency virus 1 (HIV1), Sirt1 deacetylates the viral transactivator of transcription (Tat) protein to promote the expression of the viral genome. Tat, in turn, inhibits Sirt1, leading to the T cell hyperactivation associated with HIV infection. Here, we describe the molecular mechanism of Tat-dependent sirtuin inhibition. Using Tat-derived peptides and recombinant Tat protein, we mapped the inhibitory activity to Tat residues 34–59, comprising Tat core and basic regions and including the Sirt1 deacetylation site Lys50. Tat binds to the sirtuin catalytic core and inhibits Sirt1, Sirt2, and Sirt3 with comparable potencies. Biochemical data and crystal structures of sirtuin complexes with Tat peptides reveal that Tat exploits its intrinsically extended basic region for binding to the sirtuin substrate binding cleft through substrate-like β-strand interactions, supported by charge complementarity. Tat Lys50 is positioned in the sirtuin substrate lysine pocket, although binding and inhibition do not require prior acetylation and rely on subtle differences to the binding of regular substrates. Our results provide mechanistic insights into sirtuin regulation by Tat, improving our understanding of physiological sirtuin regulation and the role of this interaction during HIV1 infection.

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.

The protein lysine deacylases of the NAD(+)-dependent Sirtuin family contribute to metabolic regulation, stress responses, and aging processes, and the human Sirtuin isoforms, Sirt1-7, are considered drug targets for aging-related diseases. The nuclear isoform Sirt1 deacetylates histones and transcription factors to regulate, e.g., metabolic adaptations and circadian mechanisms, and it is used as a therapeutic target for Huntington's disease and psoriasis. Sirt1 is regulated through a multitude of mechanisms, including the interaction with regulatory proteins such as the inhibitors Tat and Dbc1 or the activator AROS. Here, we describe a molecular characterization of AROS and how it regulates Sirt1. We find that AROS is a partly intrinsically disordered protein (IDP) that inhibits rather than activates Sirt1. A biochemical characterization of the interaction including binding and stability assays, NMR spectroscopy, mass spectrometry, and a crystal structure of Sirtuin/AROS peptide complex reveal that AROS acts as a competitive inhibitor, through binding to the Sirt1 substrate peptide site. Our results provide molecular insights in the physiological regulation of Sirt1 by a regulator protein and suggest the peptide site as an opportunity for Sirt1-targeted drug development.

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 Kyrpides, Ouzounis, Woese (KOW) domain between an α-hairpin and a β-barrel. In contrast, NusG/Spt5-KOW domains only occur in the β-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 β-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 toward the α-hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.

Periplasmic binding proteins (PBPs) are ubiquitous receptors in gram-negative bacteria. They sense solutes and play key roles in nutrient uptake. Escherichia coli's putrescine receptor PotF has been reported to bind putrescine and spermidine. We reveal that several similar biogenic polyamines are recognized by PotF. Using isothermal titration calorimetry paired with X-ray crystallography of the different complexes, we unveil PotF's binding modes in detail. The binding site for PBPs is located between two lobes that undergo a large conformational change upon ligand recognition. Hence, analyzing the influence of ligands on complex formation is crucial. Therefore, we solved crystal structures of an open and closed apo state and used them as a basis for molecular dynamics simulations. In addition, we accessed structural behavior in solution for all complexes by 1H-15N HSQC NMR spectroscopy. This combined analysis provides a robust framework for understanding ligand binding for future developments in drug design and protein engineering.

Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 degrees C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 mM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits. (C) 2021 Elsevier Ltd. All rights reserved.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antitermination (AT) is a ubiquitous principle in the regulation of bacterial transcription to suppress termination signals. In phage λ antiterminator protein Q controls the expression of the phage's late genes with loading of λQ onto the transcription elongation complex halted at a σ-dependent pause requiring a specific DNA element. The molecular basis of λQ-dependent AT and its dependence on N-utilization substance (Nus) A is so far only poorly understood. Here we used solution-state nuclear magnetic resonance spectroscopy to show that the solution structure of λQ is in agreement with the crystal structure of an N-terminally truncated variant and that the 60 residues at the N-terminus are unstructured. We also provide evidence that multidomain protein NusA interacts directly with λQ via its N-terminal domain (NTD) and the acidic repeat (AR) 2 domain, with the λQ:NusA-AR2 interaction being able to release NusA autoinhibition. The binding sites for NusA-NTD and NusA-AR2 on λQ overlap and the interactions are mutually exclusive with similar affinities, suggesting distinct roles during λQ-dependent AT, e.g. the λQ:NusA-NTD interaction might position NusA-NTD in a way to suppress termination, making NusA-NTD repositioning a general scheme in AT mechanisms.