Chemically induced protein dimerisation (CID) is a widely used approach for detecting and actuating small-molecule-protein interactions. Here, we demonstrate that CID systems can be used as building blocks for higher-order ligand-controlled protein systems. To this end, we converted classical FK506- and cyclosporine A-controlled CIDs into single-chain pseudo-allosteric proteins. These systems displayed 15- and 50-fold gains in ligand affinity compared with the parental components operating in trans. Using these fusion proteins as baits in mRNA display, we identified FN3con binding domains that recognise the holo state of each bait with subnanomolar affinity and >100-fold selectivity over the apo form. Integration of the binding domains and intramolecular CIDs into two-component enzymatic protein biosensors enabled quantification of the cognate ligands with low-nanomolar limits of detection (LODs). ELISA assays based on the developed systems displayed mid-picomolar LODs and accurately quantified FK506 and cyclosporine A in unprocessed blood samples from transplant patients with accuracy comparable to that of LC-MS/MS. This work establishes a generalisable strategy for harnessing the affinity gains of intramolecular CID systems to create gain-of-signal assays for small molecules and ions.

The large lipid transfer protein (LLTP) superfamily, characterized by a conserved LPD_N domain, plays important roles not only in lipid transport but also in diverse physiological processes, including animal development, reproduction, and immunity. However, comprehensive characterization of the LLTP gene family in cuttlefish remains limited. In this study, a total of 15 LLTP genes were identified in three cuttlefish species, Sepiella japonica, Sepia officinalis, and S. esculenta, using bioinformatic approaches. Phylogenetic analysis revealed that these genes clustered into three distinct families: VTG, Apo, and MTP. Analyses of motif patterns and gene structures revealed distinct yet conserved features among LLTP families. Chromosomal localization and synteny analyses revealed conserved organization with species-specific variations among LLTP genes. Tissue expression profiling of LLTP genes in S. japonica revealed distinct expression patterns among the three families, with VTG genes showing ovary-biased expression, whereas Apo and MTP genes exhibited broader tissue distribution patterns. Our findings advance the understanding of LLTP evolutionary diversification in cuttlefish and provide insights into their potential roles in lipid transport and other physiological processes.

Diflavin-linked disulfide oxidoreductase (DDOR) from the cyanobacterium Synechocystis sp. PCC6803 is an NADPH-thioredoxin reductase (NTR) superfamily enzyme that forms a homodimer containing two FAD molecules (FAD1 and FAD2) per monomer. FAD1 is conserved in the NTR superfamily, while FAD2 is characteristic of DDOR. Under aerobic conditions, thiol reduction of DDOR produces an apparently air-stable neutral blue semiquinone, but the respective roles of FAD1 and FAD2 in the reduction process remain unclear. To address this, spectroscopic, structural and mutational analyses were performed to investigate the contributions of the two FAD molecules. Recombinant apo- and holo-DDOR synthesised in Escherichia coli cells were separated and purified. Fluorescence titration of the apo-DDOR with FAD revealed rapid binding of two FADs. Stopped-flow analysis indicated biphasic binding of FAD to apo-DDOR. X-ray crystal structures of apo- and holo-DDORs exhibited pronounced conformational differences in the FAD-binding sites, suggesting that the first FAD may bind to the FAD1-binding site and subsequently induce formation of the FAD2-binding site. Systematic replacement of amino acid residues in the FAD2-binding site showed that mutations at the characteristic loop region bearing the GKKDSLY motif resulted in binding of three flavin species of FAD, flavin mononucleotide and riboflavin. Moreover, replacement of both Trp53 and Tyr143 at the binding site of the isoalloxazine ring of FAD2 with Ala residues yielded exclusively the apo-form. These results indicate that the loop region restricts the flavin species bound at the FAD2-binding site, and Trp53 and Tyr143 are important for FAD binding to both the FAD1- and FAD2-binding sites.

Allosteric and cryptic binding pockets represent a vast underexploited frontier in drug discovery, offering routes to biological targets historically considered undruggable. The absence of apparent binding pockets in the global conformations of apo structures, together with limitations in availability of chemical probes and experimental structures for novel targets, makes the identification of allosteric and cryptic sites particularly challenging. Despite their ability to reveal transient and otherwise inaccessible pockets, computational methods remain underutilized for allosteric and cryptic site discovery. In this perspective, we present the current state-of-the-art of computational strategies that are available in a computational scientists' toolbox to evaluate the possibility of finding such pockets when assessing a new target in a drug discovery project. Computational approaches span a spectrum from physics-based methods grounded in molecular thermodynamics to AI/ML techniques for identifying allosteric and cryptic binding sites. Physics-based approaches encompass methods operating across multiple scales, from single-conformation analyses that identify pockets based on geometry and energetics (e.g., Fpocket, SiteMap) to molecular simulations that reveal cryptic states and transient binding sites, as well as mixed-solvent and probe-based approaches (e.g., SILCS, MixMD) that delineate druggable hotspots through enhanced sampling. AI/ML methods such as PocketMiner and deep learning cofolding models (AlphaFold3, Boltz) that rapidly predict cryptic sites are discussed in the context of them being over 1,000-fold faster than classical simulations. By surveying current methods and benchmark studies, we aim to define their domains of applicability, highlight their strengths and limitations, and identify opportunities for their integration in prospective drug discovery.

The synaptic vesicle protein 2 (SV2) family is a highly conserved group of transmembrane glycoproteins sharing approximately 70% sequence identity. SV2A and SV2B are broadly distributed throughout the brain, while SV2C, the evolutionarily oldest member of the family, is primarily found in dopaminergic brain regions in the basal ganglia. Genome-wide association studies have linked SV2C to Parkinson's disease, and SV2C appears to enhance dopamine storage in synaptic vesicles, but the basis for this effect is unknown. SV2s are the target of the racetam class of antiseizure medications, and SV2A-specific ligands are widely used to treat seizures. Recently several SV2C-specific ligands have been developed, but the structural basis for ligand specificity is unclear. A better understanding of SV2C structure, function, and pharmacology could lead to better targeted therapies for epilepsy, Parkinson's disease, and other dopamine-related conditions. Here we present cryo-EM structures of apo SV2C, SV2C bound to the high affinity non-selective SV2 ligand padsevonil, SV2C bound to the SV2C-selective ligand UCB-F, and SV2A bound to the SV2A-selective ligand plosaracetam (also known as ABBV-552/SDI-118). We find that SV2C has a wider luminal opening than SV2A and SV2B, which allows for UCB-F to bind to the primary site and form favorable interactions that are not possible in the narrower primary binding site of SV2A and SV2B. We also demonstrate that UCB-F and padsevonil, but not plosaracetam, reduce dopamine release in striatal sections of mouse brain. Our biochemical experiments and structures provide insights into SV2 ligand specificity and offer a template for the rational development of therapeutics targeting SV2C.

Rhodopsin trafficking from the photoreceptor inner segment to the outer segment is essential for photoreceptor function, yet the molecular mechanism(s) regulating this process remain incompletely understood. MYO1C is an actin-based motor protein implicated in intracellular cargo trafficking. Here, we investigated its role in rhodopsin trafficking and photoreceptor cell homeostasis. In-silico docking identified a putative interaction between the MYO1C C-terminal region and the C-terminal region of rhodopsin containing the conserved VxPx ciliary trafficking motif. Biochemical studies confirmed that full-length MYO1C interacts with rhodopsin, whereas deletion of the MYO1C C-terminal domain abolished this interaction. Live-cell imaging, ciliary localization, and fluorescence recovery after photobleaching in hTERT-RPE1 cells demonstrated that the MYO1C C-terminal region is required for efficient rhodopsin trafficking, membrane localization, and ciliary targeting. In native murine rod photoreceptors MYO1C localized to both inner and outer segments. Global Myo1c deficiency in mice caused age-dependent rhodopsin mislocalization, apo-opsin accumulation and progressive retinal dysfunction, characterized primarily by reduced scotopic ERG responses and delayed a-wave recovery, beginning at 6-months, while photopic responses were relatively preserved. Rod-specific Myo1c deletion similarly caused progressive scotopic dysfunction and reduced a-wave recovery following light stimulation. In contrast, cone-specific Myo1c deletion preserved photopic function and a-wave recovery. Together, these findings identify MYO1C as an important regulator of rhodopsin trafficking and demonstrate a preferential, cell-autonomous requirement for MYO1C in maintaining rod photoreceptor homeostasis and phototransduction recovery. These findings establish a mechanistic link between MYO1C-dependent rhodopsin trafficking and age-dependent rod photoreceptor cell dysfunction.

Lipoprotein(a) [Lp(a)] is a causal risk factor for atherosclerotic cardiovascular disease composed of apolipoprotein(a) [APO(a)] covalently linked to apolipoprotein B100 (APOB). Although plasma Lp(a) concentrations are largely genetically determined, the mechanisms governing Lp(a) metabolism after particle assembly remain poorly understood. We performed a cross sectional, observational study to define the Lp(a) proteome and determine the metabolic behavior of APO(a) and APOB within circulating Lp(a) particles using integrated proteomic, kinetic, and imaging approaches. Sixteen healthy adults underwent stable isotope tracer studies with ²H₃-L-leucine and ²H₅-glycerol. Lp(a) particles were isolated by APO(a)-specific immunoprecipitation for high-resolution liquid chromatography-mass spectrometry and kinetic analyses, and extracellular vesicles (EVs) were characterized by imaging flow cytometry and super-resolution microscopy. Proteomic analysis identified 92 proteins associated with immuno-isolated Lp(a), enriched in pathways related to immunity, coagulation, and atherogenesis. Lp(a)-APO(a) exhibited a mean fractional clearance rate of 0.04 pools/day, whereas Lp(a)-APOB cleared approximately sevenfold faster (0.25 pools/day), independent of plasma Lp(a) concentration or APO(a) isoform size. Both APO(a) and APOB were detected on circulating EVs, suggesting that EVs may contribute to post-secretory Lp(a) particle remodeling. These integrated human studies demonstrate marked discordance between APO(a) and APOB turnover within circulating Lp(a) particles, challenging the prevailing assumption that both apolipoproteins behave as a single metabolic unit after Lp(a) assembly and supporting a revised model of human Lp(a) metabolism.

Synaptic vesicle glycoprotein 2C (SV2C) is a vesicular protein enriched in dopaminergic neurons of the basal ganglia that modulates dopamine storage and release, and its disruption is implicated in Parkinson's disease (PD). Despite strong genetic and pathological links to PD, there are no selective small-molecule probes for SV2C. Here, we describe an AI-enhanced virtual screening (VS) and experimental campaign that identified multiple novel chemotypes with low-micromolar affinity and marked selectivity for SV2C over SV2A and SV2B, starting from a large, general-purpose commercial library. Because no full-length high-resolution SV2C structure was available, we built a homology model using SV2A cryo-EM structures as templates and characterized its conformational landscape by molecular dynamics (MD) and Gaussian accelerated MD (GaMD) simulations in apo form and in complex with known SV2 ligands (plosaracetam, levetiracetam, brivaracetam, and padsevonil). A convolutional neural network-based scoring function (CNN_VS), retrospectively validated on a manually curated 39-ligand SV2A benchmark (r = 0.72 vs experimental pIC50), was then applied in a multi-stage funnel to 5.96 million Mcule in-stock compounds, which were sequentially filtered to 3.19 million CNS-relevant molecules before docking and rescoring. From 94 VS-prioritized candidates, 71 compounds were experimentally profiled in an orthogonal primary assay cascade combining a thermal shift assay (TSA) with a [3H]-padsevonil scintillation proximity assay (SPA), followed by Ki determination and isoform selectivity profiling for key hits. This campaign yielded 22 active molecules (31% hit rate) that naturally segregated into two categories: compounds that showed primary site competition, and compounds that did not show primary site competition with [3H]-padsevonil. A subset of competitor compounds also showed thermostabilization activity. Among these, compounds 36 and 56 emerged as particularly attractive leads, with Ki values of 24.6 uM and 3.25 uM at SV2C, respectively, and greater than 10-fold selectivity versus SV2A; compound 56 also maintained approximately 12-fold selectivity relative to SV2B. A complementary subset of SV2C-selective hits behaved as padsevonil-site competitors, providing a lead set that will serve as a template for functional characterization and future drug development for conditions that affect dopaminergic signaling. Docking analysis suggests a common binding mode anchored by conserved tryptophan residues in the SV2 pocket, a prediction independently confirmed by an unpublished SV2A-plosaracetam cryo-EM structure showing 0.76 Angstrom binding-site C-alpha RMSD relative to the SV2C model and complete conservation of the tryptophan cage. Subtle differences in the luminal domain and transmembrane region point to the structural determinants underlying isoform selectivity. Collectively, these results demonstrate that an AI-driven VS pipeline, tightly integrated with medium-throughput biophysical assays, can deliver selective SV2C binders from a general chemical library on a structurally under-characterized membrane target. The identified hits provide multiple starting points for hit-to-lead optimization and tools for probing SV2C biology and its role in PD.

The emergence of β-lactam resistance in Pseudomonas aeruginosa is driven in part by diversification of Pseudomonas-derived cephalosporinases (PDCs), particularly extended-spectrum AmpC variants that typically acquire broader substrate profiles through active-site expansion. Here, we report the structural and functional characterization of the PDC-16 variant of Pseudomonas aeruginosa β-lactamase through high-resolution crystal structures of its apo form (2.30 Å) and imipenem-bound complex (2.20 Å), complemented by kinetic and computational analyses. Contrary to the established paradigms of active-site expansion, PDC-16 exhibits a reduced active-site cavity volume (351.22 Å[3]) and decreased polarity, compared to the wild-type PDC-1 (371.74 Å[3]) and cavity expanded PDC-3 (405.43 Å[3]), arising from subtle repositioning of cavity-lining residues (Met266, Asn288, and Thr320) and Ω-loop (~S152-Y200)-mediated conformational constraints. The imipenem-bound structure captures a covalent acyl-enzyme intermediate with Ser64 and shows a conformational rearrangement of the Y200-R212 region, which adopts β3-β4 strand-like geometry in the ligand-bound crystallographic state. This observation is consistent with ligand-associated remodeling of the active-site architecture, although the crystal structures alone do not establish the dynamic mechanism underlying this transition. Consistent with these structural features, kinetic analyses revealed reduced catalytic efficiency toward ampicillin and carbapenems, while activity toward cefotaxime was comparatively preserved, indicating substrate-dependent effects associated with the constrained active-site architecture. Biochemical assays showed that avibactam produced substantially greater inhibition of PDC-16 than representative phenazine-derived compounds, underscoring that favorable computational binding does not necessarily translate into inhibitory efficacy. Together, these findings support an association between the remodeled active-site architecture and substrate-dependent catalytic behavior in PDC-16.

Although the association of lipoprotein(a) (Lp(a)) with coronary heart disease was reported over 60 years ago, and subsequent studies have identified a causal and independent role for Lp(a) in disease, many fundamental unanswered questions persist surrounding the biology of this enigmatic lipoprotein. There remain critical questions surrounding the structure and metabolism of Lp(a) and the unique biochemical properties of Lp(a) that drive its pathogenic properties in the vasculature. These questions are critical to address as we rapidly approach the availability of drugs that can specifically lower Lp(a).