Han Sun

Anisotropic NMR spectroscopy has emerged as a powerful tool for the structural elucidation of complex small molecules. Compared with residual dipolar couplings (RDCs), however, the application of residual chemical shift anisotropies (RCSAs) has remained relatively limited, largely owing to the lack of simple and efficient methods for their acquisition. Here, we report a biphasic polyarylisocyanide lyotropic liquid-crystalline (LLC) system based on single-handed helical polyisocyanides prepared by alkyne-Pd(II)-catalyzed controlled polymerization. The biphasic nature of this medium enables straightforward extraction of ΔRCSAs from a single one-dimensional 13C NMR experiment on a single sample, without requiring specialized instrumentation, devices, or postcorrection. We demonstrate the broad applicability of this approach by measuring ΔRCSAs for eight structurally diverse natural products and organic molecules. The resulting 13C ΔRCSAs are sufficiently large to enable reliable structural verification and stereochemical elucidation. Notably, the method allowed us to unambiguously resolve previously questionable 13C NMR assignments in cryptotanshinone. In addition, the chiral LLC medium exhibits pronounced enantiodiscrimination, as demonstrated for indanol enantiomers as well as significant enantiodifferentiating capabilities for several natural alkaloids. Furthermore, 19F RCSA measurements enable direct differentiation of two enantiomers even within a racemic mixture. Together, these results establish biphasic polyarylisocyanide LLCs as a simple, robust, and broadly applicable alignment platform for ΔRCSA measurements, thereby expanding the utility of anisotropic NMR for structural elucidation and chiral molecular characterization.

Stomatopod crustaceans (mantis shrimps) have extraordinary eyes with unmatched visual rhodopsin diversity, but their potential for broad color detection remains unclear. Here, we report that 17 rhodopsins, mostly from the midband eye region, are involved in color sensing and functionally diverse signaling via Gq/Gi coupling and describe the underlying mechanisms of spectral tuning. Two mid-wavelength rhodopsins preferentially absorb blue light and a majority of long-wavelength opsins absorb in the green range, but three absorb light beyond 600 nanometers, representing exquisite far red–sensitive animal rhodopsins. Several blue and red rhodopsins exhibit photoreversibility with opposite spectral shifts explained by retinal binding interactions, revealing unrecognized photochemical flexibility in stomatopod photoreception. Row-specific spectral sensitivity results from combining carotenoid filters with multiple individually tuned rhodopsins, altogether extending broad vision to the far-red spectrum.

BAX is a critical regulator of the apoptosis cascade and its abnormal activation contributes to the development of disease including cardiovascular and neurodegenerative disorders. Therefore, BAX represents an attractive target for the pharmacological inhibition of pathological cell death. Based on a virtual screen, we identified and modified several EGFR kinase inhibitors with potential activity against BAX. Systematic modifications of the dacomitinib and gefitinib core structure were performed, allowing us to elucidate how structural modifications influence biological activity and leading to a comprehensive structure-activity relationship (SAR) on BAX inhibition. The developed series has favorable drug-like properties and improved BAX inhibitory activity in vitro, in cells, and in human induced pluripotent stem cell-derived brain organoids, with significantly reduced EGFR kinase activity. This series therefore represents a promising starting point for the development of improved and potentially therapeutic BAX inhibitors.

Mechanosensitive channels are central to cellular responses to membrane tension, yet the structural basis of their gating remains incompletely understood. Here, we determine the structures of wild-type and Gly22→Ser (G22S) mutants of MscL from Escherichia coli ( Ec MscL) by cryogenic electron microscopy (cryo-EM) in peptide-based lipid nanodiscs and complement them with solid-state nuclear magnetic resonance (NMR) measurements in liposomes to capture their dynamics in a native-like membrane environment. The cryo-EM structures reveal a closed conformation, whereas analysis of the low-threshold G22S mutant by NMR uncovers widespread conformational changes in both cytoplasmic and periplasmic regions. These data indicate enhanced dynamics and conformational heterogeneity in the mutant, revealing the early transitions from the closed toward the open state. Together, our results establish a synergistic framework integrating cryo-EM and NMR to resolve both structure and dynamics of mechanosensitive channels, and identify lipid-protein interactions as key determinants of MscL gating and mechanosensitivity. Our study further provides a quantitative benchmark for computational investigations of mechanogating and lays the foundation for the rational design of channels with tunable gating kinetics.

The TREK two-pore domain potassium channels regulate membrane excitability and respond to diverse physiological stimuli. Single-site mutations of TREK are associated with neurodevelopmental disorders and can cause pathological channel hyperactivation. Here, using the OneOPES enhanced-sampling molecular dynamics framework, we characterize the energetics of conformational transition along a predefined pathway connecting the up and down states of TREK-2, together with initial conformational rearrangements of the selectivity filter under physiological stimuli and disease-mimicking conditions. The simulations reveal a shared energetic axis coupling the lower helices, proximal C-terminus, and selectivity filter to govern channel gating. Predicted conformational effects of a disease-mimicking mutation were validated electrophysiologically using a conformation-sensitive TREK-2 inhibitor. Together with our previous unbiased MD simulations, these results establish an energetic framework for TREK-2 regulation and dysfunction, complementing prior structural and mechanistic studies while highlighting the potential and methodological challenges of advanced enhanced-sampling approaches for resolving complex conformational landscapes of ion channels.

Scorpion toxins block potassium channels, disrupting cellular excitability and causing symptoms such as pain, muscle spasms, or paralysis. Here, we use an integrated structural biology approach to uncover the binding mode of the scorpion toxin charybdotoxin (CTX) to the MthK channel, a model system for human large-conductance potassium (BK) channels. Cryo-EM defines the overall architecture of the MthK–CTX complex, while complementary solution- and solid-state NMR experiments identify key binding residues and show that toxin engagement alters the selectivity filter (SF) ion configuration without rearranging the filter itself. NMR and MD simulations further reveal an anchoring lysine residue stably inserted into the SF, while other contacts undergo fast NMR timescale dynamics. Together, these findings explain how CTX-like toxins maintain exceptionally high affinity while tolerating binding across multiple K⁺ channel subtypes, paving the way for site-specific extracellular modulation.

Macrocyclic peptides are emerging as a powerful therapeutic modality owing to their potential oral bioavailability and capacity to engage targets long considered undruggable. The introduction of aromatic heterocycles into macrocycles further expands this structural space by imparting distinct conformational preferences. However, accurate determination of the solution-state structures and dynamics of the resulting molecules remains challenging. Here, we integrate complementary isotropic and anisotropic NMR observables with enhanced sampling simulations and density functional theory (DFT) calculations to systematically investigate aryl- and heterobiaryl-containing cyclic peptides in different solvent systems. Our results based on residual dipolar coupling (RDC) measurements reveal conformational dynamics of macrocycles and significantly extend the knowledge gained by conventional NMR analysis based on nuclear Overhauser effects (NOEs) by more faithfully capturing the solution-state ensembles. Our methodology enables conformational analysis in both fast- and slow-exchange regimes on the NMR time scale. Notably, up to three interconverting backbone conformers at the DFT level are required to fully reconcile the experimental data for each ring system. We identify aryl and heterobiaryl motifs as structural elements governing the conformational landscape in our systems, modulating the populations of multiple thermodynamically accessible states characterized by distinct intramolecular hydrogen-bonding networks and unusual backbone geometries. Together, this integrative NMR-computational framework provides a precise and general strategy for resolving complex conformational ensembles of macrocycles, which should inform the objectives for synthetic modification and pave a way for the structure-based rational design of next-generation peptide therapeutics.

Providing immediate access for functional proteins inside living cells would unlock unprecedented control over cellular processes; however, commonly used endocytic delivery suffers from endosomal trapping and degradation. One of the most powerful non-endosomal delivery methods uses cell surface anchored cell penetrating peptide (CPP)-additives that allow proteins to enter cells directly. Nevertheless, the underlying molecular mechanism involved in direct entry via crossing the cell membrane (protein translocation through the cell) and the major driving forces remain controversially discussed. Here, we provide a stepwise molecular picture on how CPP-additives enable uptake of protein cargoes through direct membrane translocation. CPP-additives accumulate on the cell surface in nucleation zones, locally hyperpolarizing the membrane, and induce transient water pores that allow selective CPP-protein entry without compromising membrane integrity. These fundamental mechanistic insights provide a firm basis for rationally optimizing delivery strategies using highly cationic CPPs, ultimately resulting in innovative and smart protein delivery strategies to advance therapeutic protein applications. Abstract Figure:

The TREK subfamily of two-pore domain potassium (K _2P ) channels are essential regulators of membrane excitability, and their activity is modulated by a wide range of physiological stimuli, including phosphorylation and membrane stretch. Single-site mutations in this subfamily have been identified in patients with FHEIG symptom (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth), where they cause pathological channel hyperactivation. Using OneOPES framework, which unifies multiple enhanced-sampling molecular dynamics strategies, we provide a detailed energetic characterization of the TREK-2 conformational landscape. Our simulations uncover a unifying mechanism in which coupling between the lower transmembrane helices, the proximal C-terminal domain, and selectivity filter stability governs channel gating under different physiological stimuli, as well as disease-mimicking conditions. The predicted conformational effects of an FHEIG syndrome-associated mutation were further validated by electrophysiological measurements using a conformation-sensitive TREK-2 inhibitor. Together, these results establish an energetic framework for TREK-2 regulation and dysfunction and provide a foundation for structure-based drug discovery targeting the K _2P TREK channel family.

Covalent inhibitors and chemical probes targeting ligandable cysteine residues have emerged as powerful tools for drug discovery and proteomics. In this study, we introduce vinyl phosphonamidates (VPAs) as a novel class of latent cysteine electrophiles and assess their reactivity, selectivity, and potential for developing covalent inhibitors. Compared to well-established chloroacetamide and acrylamide electrophiles, VPAs exhibit a significantly lower intrinsic reactivity toward the model thiol glutathione. Moreover, VPA-derived covalent fragments displayed only very limited nonspecific reactivity in human cell lysate. Encouraged by these results, we developed VPA-functionalized derivatives of the FDA-approved covalent inhibitors Afatinib and Ibrutinib and evaluated their ability to engage the target protein by gel-based and mass spectrometry-based activity-based protein profiling (ABPP). Compared to commonly employed Michael acceptor-based electrophilic groups, VPA-functionalized drug ligands displayed significantly less off-targets while maintaining inhibitor efficiency. Furthermore, we leveraged the modular nature and accessibility of VPAs to develop a bifunctional proteolysis targeting chimera (PROTAC) for targeted protein degradation. The demonstrated selectivity and modularity, as exemplified by the incorporation of various ligands on the phosphorus O-substituent, of the vinyl phosphonamidate group as a cysteine-directed electrophile highlight its ability to expand the chemical space in the development of covalent inhibitors with a favorable proteome-wide reactivity profile.