JACS Au

Rational design of covalent protein-labeling reagents in complex biological environments requires a molecular-level understanding of how the protein microenvironment governs chemical reactivity; yet, such mechanistic details remain inaccessible to experimental methods alone. In living neurons, Ligand-Directed Acyl Imidazole (LDAI) chemistry has been used to label AMPA receptors as a traceless, affinity-based protein labeling method. Although LDAI labeling reagents have been optimized in the lab, the atomic details of their interactions with the protein and the underlying mechanism remain elusive. In this work, we combined Quantum Mechanical (QM) calculations and molecular dynamics (MD) simulations to propose a detailed reaction mechanism for AMPAR labeling by LDAI reagents and to clarify how the protein microenvironment governs reactivity. Although Lys residues are usually protonated at physiological pH and therefore less nucleophilic in water, our QM results show that Lys labeling is energetically more favorable than competing reactions with Ser or water. MD simulations reveal that PFQX ---the LDAI reagent precursor--- binds dynamically to the GluA2 AMPAR as an antagonist, inducing conformational changes that reshape the local environment of the acyl imidazole (AI) warhead, underscoring that ligand identity strongly affects labeling outcomes. We also identified intra and intermolecular hydrogen bond networks that may contribute to further immobilize and pre-organize the LDAI reagent. Moreover, the probe's chemical nature shapes its interactions with the Ligand Binding Domain (LBD), offering a plausible rationale for the previously experimentally observed ligand-dependent fluorescent response. Taken together, our results establish design principles for exploiting the reagent geometry and binding pocket hydrogen-bonding networks for the rational design of LDAI reagents.

Protein phosphatase 2A containing the B56{delta} regulatory subunit (PP2A-B56{delta}) is a critical signaling enzyme whose dysregulation is associated with cancer, neurodegenerative disorders, and Jordan's syndrome, a severe intellectual disability disorder caused by mutations in B56{delta}. Unlike other PP2A holoenzymes, PP2A-B56{delta} is regulated through a unique dual autoinhibition mechanism in which the N- and C-arms occlude the catalytic site while a substrate-mimicking short linear motif (SLiM) blocks the substrate-binding pocket. Although disease-associated mutations have been shown to alter enzyme activity, the molecular mechanism underlying activation of PP2A-B56{delta} and the effects of pathogenic mutations remain poorly understood. Here, we combined cryo-electron microscopy (cryo-EM), enhanced-sampling molecular dynamics (MD) simulations, Markov state model (MSM) construction, and transition-state analysis using Transition State identification via Dispersion and vAriational principle Regularized neural networks (TS-DAR) to characterize the conformational landscape of the disease variant E198K. Our cryo-EM analysis identified two distinct structures of E198K: an inactive closed-form with the N/C-arms resolved and an active loose-form in which the N/C-arms become highly flexible and could not be fully resolved. These structures therefore established that activation is governed by conformational changes of the N/C-arms but did not reveal the underlying mechanism. Starting from the inactive closed-form, we generated over 1,600 trajectories with an average length of 1,260 ns combined for E198K and wild-type (WT) PP2A-B56{delta}. TS-DAR identified four metastable states and two major activation pathways connecting inactive and active conformations. We found that activation occurs through progressive loosening of the N/C-arm interface while maintaining the overall holoenzyme architecture, rather than a complete opening of the interface. This mechanism exposes both the catalytic site and substrate-binding pocket. Comparison of E198K and WT revealed that the disease-associated mutation shifts the conformational equilibrium toward active states while leaving the transition-state ensemble largely unchanged. Mechanistically, E198K disrupts a salt-bridge network and weakens interactions between the internal loop and the C-arm that normally stabilize active-site occlusion. The resulting increase in C-arm mobility promotes active-site exposure and explains the elevated catalytic activity of the mutant. Together, these findings establish a previously uncharacterized activation mechanism for PP2A-B56{delta} and provide an atomic-level explanation for how the pathogenic E198K mutation allosterically promotes holoenzyme activation.

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

Chemically similar post-translational modifications can mediate distinct biological functions, but how proteins distinguish between them remains unclear. Sulfotyrosine (sTyr) and phosphotyrosine (pTyr) exemplify this problem because they have similar sizes, local geometries, and electrostatic properties but function in different biological contexts. Here, we used the monoclonal antibody PSG2, which recognizes sTyr independently of the surrounding peptide sequence, to examine how a protein distinguishes these modifications. The crystal structure of PSG2 bound to an sTyr-containing peptide revealed a deep electropositive pocket with no modeled water molecules in direct contact with the sulfate group. Gas-phase density functional theory calculations favored pTyr over sTyr, showing that direct protein-ligand interactions alone are insufficient to explain PSG2 selectivity. Explicit first-shell hydration calculations showed that pTyr has a larger desolvation penalty than sTyr, and accounting for this difference reversed the calculated energetic order. Isothermal titration calorimetry showed favorable enthalpic and entropic contributions to sTyr binding, whereas no detectable heat signal was observed for pTyr. These results show that PSG2 distinguishes sTyr from pTyr through the balance between direct protein-ligand interactions and ligand desolvation.

Investigating the conformational dynamics of intrinsically disordered proteins (IDPs) is essential to understanding how their structural heterogeneity underlies function and how their dysregulation contributes to diseases. Here, we utilized an MspA nanopore-based approach for studying the conformational dynamics and interactions of IDPs at the single-molecule level. The platform was demonstrated using the intrinsically disordered transactivation domain of tumor suppressor p53 (p53-TAD), one of the important proteins in cancer biology. We showed that MspA can stably capture p53-TAD and resolve up to six distinct current states with frequent interconversions, revealing a rich conformational landscape. The nanopore also detected the effect of a cancer-associated double mutational variant, N29K/N30D. Combining experiments with steered molecular dynamics simulations, we showed that the mutant sampled compact conformational states more frequently than wild type, consistent with previous NMR studies. Importantly, the MspA platform enabled direct monitoring of E3 ligase MDM2 binding to p53-TAD and resolved how this interaction is inhibited by anti-cancer compound epigallocatechin gallate (EGCG). Notably, EGCG stabilizes one of the six states sampled by p53-TAD, providing a mechanistic explanation for its inhibitory effect. Together, these findings demonstrate the promise of the nanopore platform for label-free monitoring of IDP conformational dynamics, modulation, binding and inhibition at single-molecule resolution.

The non-amyloid-{beta} component (NAC) region of the Parkinson's-associated protein -synuclein plays a key role in its pathogenic aggregation, motivating the development of molecules that target this critical region. Here, we show that a minimal NAC-derived motif, 66VGGAVVT72, can be reprogrammed through backbone engineering to modulate -synuclein aggregation. Backbone thioamide substitution of this peptide enhances its interactions with -synuclein fibrils and accelerates aggregation, whereas N-methylation disrupts {beta}-sheet hydrogen bonding and inhibits fibrillization. Strikingly, combining these modifications yields hybrid peptides that inhibit the fibrillization of full-length -synuclein at sub-stoichiometric concentrations. Consistent with in vitro results, these backbone-modified peptides can also reduce seeded -synuclein aggregation in cells. These results establish that minimal amyloidogenic sequences can be systematically tuned from aggregation promoters to inhibitors through backbone-level perturbations, particularly thioamide incorporation.

Calcium oxalates are naturally occurring minerals, with the monohydrate form CaC2O4xH2O (COM) being the most stable. COM contains two crystallographically different water sites. We investigate the details of water internal dynamics in the high and low temperature phases of COM using 17O central transition solid-state NMR line shapes, as well as laboratory and rotating frame relaxation rates. The measurements were performed either under static or magic angle spinning conditions and in a wide temperature range from 343 to 180 K. The combination of all measurements allows for precise constraints on motional mechanisms, rate constants, and amplitudes of motions. The high temperature phase is dominated by large-angle fluctuations with an amplitude of about 100 degrees, identical in both sites. During the phase transition between 323 to 300 K, these large-angle jumps freeze out in one of the water sites, while remaining active in the other. In the low temperature phase from 280 to 180 K, small-angle fluctuations of 2-8 degrees in amplitude dominate the relaxation. Transverse relaxation rates also point to the existence of a very slow collective rocking motion down to about 220-200 K.

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

Reading both physical and chemical properties of a microenvironment from a single fluorophore remains a challenge. Here we demonstrate that two coexisting molecular equilibria within one near-infrared cyanine, CyC4, encode two mechanistically distinct ratiometric reporting channels. A meso-amino group and a pendant carboxylate form a tunable intramolecular hydrogen bond that toggles the dye between closed (700 nm) and open (780 nm) emissive conformers. Time-dependent density functional theory (TD-DFT) calculations show that the hydrogen bond raises the LUMO and blue-shifts the emission, establishing the 700/780 emission ratio as a local reporter of hydrogen bonding and polarity. Independently, the chromophore self-associates under crowding- and cosolvent-rich conditions into an aggregate with a blue-shifted, H-type absorption signature near 530-540 nm and a distinct emission near 610 nm upon 540 nm excitation. The intensity of this aggregate band relative to the monomer emission (Ra) serves as a ratiometric reporter of crowding and self-association. Because the two channels arise from distinct molecular equilibria (intramolecular hydrogen bonding vs. intermolecular self-association) they are largely decoupled: a glycerol titration series confirms that the self-association channel (Ra) can be moved while the hydrogen-bonding channel stays essentially fixed. Applied to protein-PEG biomolecular condensates, the two ratios move oppositely with increasing salt, showing that the interior's chemical (polarity, hydrogen bonding) and physical (packing, self-association) environments co-vary across the salt series; a single CyC4 measurement thereby maps this coupled microenvironment, providing a general strategy for multiparametric, ratiometric sensing of crowded microenvironments.

Residual dipolar couplings (RDCs) obtained via magnetic field-induced alignment offer a powerful, media-free approach for the structural analysis of biomolecules. However, their detection in short, fast-tumbling nucleic acids remains elusive at conventional magnetic fields due to insufficient alignment and sensitivity. Here, we demonstrate the direct observation of these RDCs at 1.3 GHz in a 14-mer hairpin fragment derived from an HIV-1 Vif-targeting aptamer. The 1JNH scalar couplings of imino protons were measured at fields ranging from 600 MHz to 1.3 GHz. While the coupling constants remained invariant between 600 and 900 MHz, a clear deviation was exclusively captured at 1.3 GHz for all base-paired stem residues, demonstrating the first media-free detection of field-induced RDCs in a short RNA of this size. This breakthrough arises from a synergistic B07/2 scaling, combining enhanced alignment ({propto} B02) and sensitivity ({propto} B03/2). These RDCs showed excellent agreement with the NOE-derived structure. Additionally, the flexible loop residue G8 exhibited no detectable RDC, but displayed a field-dependent TROSY/anti-TROSY intensity inversion at 1.3 GHz, reflecting an unusual 1H chemical shift anisotropy (CSA) tensor that corroborates the local base-packing environment. Our findings highlight 1.3 GHz NMR as an indispensable tool for the structural analysis of short RNAs.

The enzymatic removal of sulfate groups regulates processes ranging from steroid metabolism to carbohydrate degradation. Most sulfatases belong to the S1 family, whose members use a co-translationally installed formylglycine residue to hydrolyse sulfate esters. Arylsulfamates are potent covalent inhibitors of aryl and steroid sulfatases, including the clinical steroid sulfatase inhibitor Irosustat, yet the structure and stability of the inhibited complex remain unresolved. Arylsulfamates and carbohydrate sulfamates do not covalently inhibit many S1 carbohydrate sulfatases despite conservation of their sulfate-binding sites and formylglycine residue. Using enzyme kinetics, X-ray crystallography, molecular dynamics simulations and density functional theory calculations, we define the basis of these contrasting behaviours. High-resolution structures of the Pseudomonas aeruginosa arylsulfatase PaAtsA treated with two arylsulfamates reveal a long-lived tetrahedral, O-linked -hydroxysulfamate adduct attached to formylglycine. Molecular simulations show that replacing sulfate with sulfamate disrupts the favourable Ca2+-oxyanion interaction and alters ligand binding geometry. The permissive hydrophobic binding site of PaAtsA accommodates this rearrangement while retaining a trajectory compatible with nucleophilic attack. By contrast, in the Bacteroides thetaiotaomicron carbohydrate sulfatase BT16363S-Gal, sulfate-to-sulfamate substitution weakens binding and displaces the sulfamate from a reactive pose near the catalytic nucleophile due to a restrictive active site with conserved sugar binding. These findings define the structure and persistence of the arylsulfamate-derived covalent intermediate and explain why sulfamate warheads are tolerated by aryl sulfatases but not carbohydrate sulfatases.

The navigational prowess of migratory birds is thought to arise from light-dependent radical-pair chemistry in cryptochrome 4 (CRY4), yet the slow structural transitions that couple photochemistry to signaling remain elusive. Here, we combine temperature-controlled steady-state UV-visible spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate the photochemical and conformational dynamics of pigeon CRY4 (ClCRY4). Steady-state measurements at 5-25 {degrees}C reveal that lower temperatures slow FAD photoreduction and prolong the FAD neutral semiquinone signaling state. This occurs without a solvent kinetic isotope effect, implicating a conformational change rather than proton transfer as the rate determining step in FAD neutral semiquinone formation. Simultaneous HDX-MS under blue-light exposure identifies protection near the FAD-binding site and C-terminal region. To enhance sensitivity, we developed a pump-probe HDX-MS approach at 10 {degrees}C. This reveals eight peptides (within the phosphate-binding loop, protrusion motif, electron-transfer-chain loops and C-terminal tail) that exhibit rapid ([≤]10 s) and sustained light-induced protection, delineating early conformational rearrangements as a prerequisite for FAD neutral semiquinone accumulation. The findings of slower onset HDX protection as well as a bimodal pattern of deuterium uptake in the phosphate-binding loop further identify a local redistribution of conformational substates on the time scale of the accumulation of the signaling species. Site specific mutagenesis within the CTT supports the findings, which lead to a model in which blue light triggers rapid clamping down of protein near the two regions of spin pair separation, followed by a rate limiting closure of a surface loop. The resolution of time-dependent structural transitions that follow photoactivation of CRY4 resolves the interface between quantum radical-pair formation and classical conformational changes, while providing an enhanced structural framework for the molecular events that underlie avian magnetoreception.

The rotary motor F1-ATPase has been extensively studied as a model molecular machine, yet rational engineering of its catalytic activity remains challenging because ATP hydrolysis is regulated by long-range intersubunit allostery and large conformational transitions. Here, we developed a homolog-guided engineering strategy to increase the maximum rotation rate of the thermophilic Bacillus PS3 F1-ATPase (TF1). Candidate mutation sites were first identified by comparing TF1 with the homologous enzymes bovine mitochondrial F1 (bMF1) and Paracoccus denitrificans F1 (PdF1), both of which exhibit higher maximum rotation rates than TF1. Systematic exploration of these sites identified four activity-enhancing hotspots, followed by focused hotspot exploration and machine-learning-assisted prioritization of combinatorial mutants. The best mutant, TF1({beta}Y313L/{beta}E332S), exhibited a 1.8-fold higher maximum rotation rate than TF1(WT) while retaining its functional thermostability. Interestingly, activity-enhancing substitutions were not limited to the residues conserved in both bMF1 and PdF1, indicating that the bMF1-PdF1 consensus substitutions effectively identify activity-enhancing hotspots rather than uniquely defining the optimal amino acid. Machine-learning-assisted exploration efficiently prioritized highly active mutants, although the predictive performance was limited by the relatively small training dataset and epistatic interactions among mutations. Kinetic and structural comparisons further provided mechanistic insights into the enhanced catalytic activity of the engineered mutant. Together, these results establish a practical strategy for engineering complex molecular motors by combining homolog-guided hotspot identification with focused hotspot exploration.

Contemporary RNA-binding ligand collections are biased toward aromatic scaffolds, although it remains unclear whether this over-representation reflects an intrinsic requirement for productive RNA recognition or historical discovery bias. Here, using a modular "host-guest" ligand design strategy targeting the env8 cobalamin (Cbl) riboswitch, we established a common molecular framework to directly evaluate whether aromaticity is fundamentally required for RNA binding. We synthesized a focused series of cyclic aliphatic {beta}-axial Cbl derivatives, expanding the ligand library and enabling matched-pair comparisons to isolate the contribution of aromaticity to molecular recognition. Aliphatic ligands supported high-affinity RNA binding and regulatory activity comparable to aromatic analogues, with several derivatives exhibiting equal or greater affinity than their matched aromatic counterparts. Structural analyses revealed that aromatic and aliphatic ligands engage the same cryptic RNA binding site through distinct modes of molecular recognition, including nucleobase {pi}-stacking and alternative van der Waals packing arrangements. Machine learning analyses further demonstrated that the physicochemical features associated with affinity extend beyond aromaticity itself and instead reflect a broader combination of shape, surface, heteroatom, and electronic properties. Together, these findings demonstrate that high-affinity RNA binding can arise from multiple structural and physicochemical solutions, suggesting that aromaticity is not uniquely privileged as a strategy for RNA-targeted ligand design and supporting broader exploration of underrepresented RNA-binding chemotypes.

Weak, reversible interactions underpin biomolecular recognition, and single-molecule fluorescence (smF) imaging techniques can provide unprecedented insight into those biological processes. Unfortunately, such studies often require micromolar concentrations of fluorophore-labeled biomolecules, which is beyond the accessible range of conventional smF microscopies. Here, we describe a surface-functionalization method based on cloud-point polyethylene glycol (PEG) grafting that enables widefield smF microscopy measurements at micromolar concentrations without the use of nanophotonic devices. Using conventional total internal reflection fluorescence (TIRF) microscopy, we detected single-molecule fluorescence resonance energy transfer (smFRET) from surface-tethered, donor-labeled target molecules with up to 8 micromolar concentrations of freely diffusing, acceptor-labeled analyte molecules in the background--two orders of magnitude higher than typical studies in the literature. Weak, DNA-hybridization and protein-RNA binding equilibria were measured across micromolar range titrations. Together with advances in high-background data analysis, the robust method presented here enables kinetic and thermodynamic analyses of weak biomolecular interactions, especially those limited by nonspecific adsorption and high fluorescence backgrounds, using only standard smF instrumentation.

Hepatitis B virus (HBV) infection depends on coordinated capsid assembly and virion production. A hydrophobic pocket at the intradimer interface of the HBV core protein has been linked to secretion phenotypes and shown to bind small molecules, but its interaction landscape remains poorly defined. Here, we combine atomistic molecular dynamics (MD) simulations, fragment mapping, pharmacophore modeling, and biophysical binding assays to characterize this pocket and identify ligands with binding modes that extend beyond the known pocket. This workflow narrowed an initial library of approximately 4.7 million compounds to eight candidates for experimental evaluation by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). Of these eight, compound B3 showed detectable STD NMR signals, concentration-dependent SPR binding, and an MD-supported binding mode that retained hydrophobic-pocket anchoring while also extending toward the spike-proximal loop. Together, these results illustrate how dynamic fragment mapping can identify ligand candidates that engage broader interaction landscapes at protein-interface pockets.

Direct visualisation of drug-target engagement within living cells remains a major challenge. Here we develop a cell-permeable fluorogenic analogue of the phosphodiesterase 4 (PDE4) inhibitor rolipram (MAN193), generated by conjugation to fluorescein diacetate. Following intracellular activation, the probe functions as a near-neutral PDE4 antagonist and enables direct visualisation of endogenous PDE4 populations. Using advanced fluorescence spectroscopy imaging approaches, including molecular brightness analysis and fluorescence anisotropy imaging, we demonstrate rolipram-displaceable binding of the analogue to cytosolic PDE4 and resolve thesubcellular distribution of binding sites. We further show that the probe enables visualisation of untagged PDE4 at focal adhesion complexes in cardiomyocyte-like cells. Together these findings establish fluorogenic drug conjugation combined with quantitative spectroscopy imaging as a generalisable strategy to map intracellular drug-target engagement with subcellular resolution, providing an effective framework for interrogating the spatial pharmacology of small molecules in living systems.

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.

Cereblon (CRBN) is widely used in targeted protein degradation, but its ligand space has remained dominated by a narrow set of cyclic imide chemotypes. Here, we show that the accessible CRBN ligand space extends substantially beyond this canonical space. A high-throughput screen of > 40,000 compounds, followed by orthogonal biophysical validation, X-ray crystallography and SAR analyses, identified several chemically distinct ligand classes. These include linear acetyl-based motifs, a phthalide-derived scaffold, steroidal compounds, and a range of bicyclic ligands. They engage CRBN through distinct recognition modes, several of which deviate from the canonical hydrogen-bonding pattern. Steroidal scaffolds were particularly notable: cortisone binds the human CRBN thalidomide-binding domain with an affinity comparable to thalidomide, with its A-ring occupying the tri-tryptophan pocket in a glutarimide-like orientation despite lacking the canonical imide NH donor. SAR within this series showed substantial tolerance for chemical modification and scaffold simplification, raising the possibility that endogenous steroidal metabolites may contribute to the physiological ligand landscape of CRBN. Bicyclic lactams additionally provided synthetically accessible scaffolds with tunable affinity and promising sites for linker attachment. Across the identified ligand classes, none of the tested representatives induced detectable degradation of canonical CRBN neosubstrates, and several showed largely clean proteomic profiles. Together, these findings broaden the chemical, mechanistic and potential physiological landscape of CRBN recognition and provide diverse starting points for alternative, potentially neosubstrate-sparing CRBN recruiters.