Protein prenylation is a critical post-translational modification that controls many cancer-related signalling pathways and represents an important therapeutic target currently lacking effective pharmacological agents. Here, we establish the fluorescent prenyl diphosphate analogue MANT-O-GPP as a multipurpose probe for simultaneous analysis of ligand binding and catalysis in geranylgeranyltransferase I (GGTaseI) and farnesyltransferase (FTase). Using a tryptophan-to-MANT-O-GPP FRET assay, we found that MANT-O-GPP bound to GGTaseI and FTase with high affinity and reported occupancy of the isoprenoid donor site, as confirmed by displacement with the native substrates. In parallel, a FRET-based activity assay employing CFP-tagged protein substrates enabled direct monitoring of prenyl transfer and product formation. Notably, the known inhibitor L-778123 blocked catalysis without displacing MANT-O-GPP, demonstrating that the combined platform distinguishes prenyl-site competitors from inhibitors acting through the adjacent protein-substrate region. This fluorescence-based system provides a practical, mechanistically informative, and high-throughput-compatible platform for prenyltransferase inhibitor discovery.

Flexible proteins populate heterogeneous conformational ensembles that are essential for their function. Small-angle x-ray scattering (SAXS) is widely used to study protein structure in solution and to characterize conformational heterogeneity. Yet, extracting ensemble information from SAXS remains challenging because many distinct conformational distributions can give rise to the same experimental scattering profile. The ensemble optimization method (EOM) addresses this by selecting SAXS-consistent ensembles from large conformational pools, but commonly used pool-generation strategies rely on user-defined flexible regions and may include energetically unfavorable conformations. Here, we implement BioEmu, a generative deep-learning biomolecular emulator, as a sequence-based, physically informed prior for generating conformational pools in SAXS ensemble analysis. Using the flexible protein CLIC5 as a proof-of-concept, BioEmu sampled compact and rare elongated, interface-exposed conformations ab initio. The unweighted BioEmu pool average did not reproduce the experimental scattering, indicating that EOM-guided selection remained necessary. Convergence analysis across five distinct flexible protein systems showed that EOM fit quality plateaus at ~1000 BioEmu conformers pool, underscoring the feasible computational cost of this approach. Finally, we demonstrate the generalizability of BioEmu implementation for SAXS analysis on a diverse cohort of 20 SASBDB entries, ranging from intrinsically disordered to multi-domain proteins. Together, our results support BioEmu as a scalable, sequence-based prior for EOM-based SAXS modeling, while underscoring that selected ensembles should be interpreted as SAXS-consistent models rather than unique representations of equilibrium populations.

BACKGROUND:Congenital short-QT syndrome (SQTS) is a genetic disorder characterized by short QT interval on electrocardiography (ECG) and a high risk for malignant ventricular tachyarrhythmias. OBJECTIVES:The aim of this study was to describe a new variant in the SQTS-associated gene SLC4A3 at the molecular and clinical levels. METHODS:Using whole-exome sequencing, a novel missense variant in SLC4A3 was identified, encoding for the cardiac anion exchanger 3. The mutant was characterized using computational structural modeling and functional transport studies in human embryonic kidney 293 cells. Patients were assessed using resting ECG, 12-lead Holter recordings, and a novel diagnostic test termed here the Ippon test. RESULTS:A novel heterozygous SLC4A3 variant (p.R1016G) was detected in a family with 6 cases of sudden cardiac death and a case of documented polymorphic ventricular tachycardia in 5 generations. Functional analyses in human embryonic kidney 293 cells revealed gain of function rather than the loss of function expected on the basis of previously reported SQTS-associated SLC4A3 variants. Although affected family members exhibited shorter corrected QT intervals on resting ECG compared with nonaffected members (360 ± 20 ms vs 380 ± 30 ms; P = 0.0068) and 12-lead Holter monitoring (350 ± 20 ms vs 380 ± 30 ms; P = 0.0013), significant overlap existed. The sudden heart rate deceleration provoked by the Ippon test revealed that the QT interval in carriers failed to prolong in response to the sudden bradycardia, resulting in inappropriately short corrected QT intervals, leading to a better distinction of affected from nonaffected patients (340 ± 30 ms vs 370 ± 10 ms, respectively; P = 0.0003). CONCLUSIONS:SLC4A3 p.R1016G is a novel SQTS-associated variant associated with a gain-of-function effect. The Ippon test is a new provocation maneuver that identifies SQTS variant carriers with high diagnostic accuracy.

Central centrifugal cicatricial alopecia (CCCA) is the most common form of primary scarring alopecia in women of African descent, typically characterized by progressive hair loss originating at the vertex of the scalp. Although genetic susceptibility has been implicated in the pathogenesis of CCCA, only 1 gene (PADI3, encoding peptidyl arginine deiminase 3) has been thus far associated with CCCA. This study aimed to broaden our understanding of the genetic basis of CCCA by analyzing whole-exome sequences from 75 patients with clinically and histologically confirmed CCCA. We identified 9 pathogenic heterozygous variants in PADI3, including, to our knowledge, 4 previously unreported missense variants, all predicted to disrupt protein function. Functional analyses revealed reduced expression, abnormal intracellular localization, and diminished enzymatic activity in cells transfected with constructs expressing the PADI3 variants. More interestingly, pathogenic variants were identified in 2 additional genes, S100A3 and TCHH, which encode the main substrates of PADI3, S100 calcium-binding protein A3 and trichohyalin. Both proteins play critical roles in hair shaft integrity. The S100A3 variant was found to cause reduced citrullination by PADI3, whereas TCHH variants altered intracellular localization and resulted in significantly reduced expression of the protein. These findings provide further insights into disease mechanisms and may inform future strategies for genetic testing and targeted therapies.

Epidermolysis bullosa simplex (EBS) refers to a heterogeneous group of inherited skin disorders characterized by blister formation within the basal cell layer. The disease is characterized by marked variations in phenotype severity, suggesting co-inheritance of genetic modifiers. We identified three deleterious variants in HMCN1 that co-segregated with a more severe phenotype in a group of 20 individuals with EBS caused by mutations in KRT14, encoding keratin 14 (K14). HMCN1 codes for hemicentin-1. Protein modeling, molecular dynamics simulations, and functional experiments showed that all three HMCN1 variants disrupt protein stability. Hemicentin-1 was found to be expressed in human skin above the BMZ. Using yeast-2-hybrid, co-immunoprecipitation, and proximity ligation assays, we found that hemicentin-1 binds K14. Three-dimensional skin equivalents grown from hemicentin-1-deficient cells were found to spontaneously develop subepidermal blisters, and HMCN1 downregulation was found to reduce keratin intermediate filament formation. In conclusion, hemicentin-1 binds K14 and contributes to BMZ stability, which explains the fact that deleterious HMCN1 variants co-segregate with a more severe phenotype in KRT14-associated EBS.

Human cis-prenyltransferase (hcis-PT) synthesizes long-chain isoprenoids essential for N-linked protein glycosylation. This heteromeric complex comprises the catalytic subunit DHDDS and the regulatory Nogo-B receptor (NgBR). Although NgBR dramatically enhances DHDDS activity, the molecular basis for this allosteric regulation remains unclear. Here, we combined crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics simulations, and network analysis to uncover the structural dynamics and communication pathways within hcis-PT. By solving the apo structure of hcis-PT, we reveal only a localized flexibility at the active site and the NgBR C-terminus. However, HDX-MS demonstrated widespread substrate-induced stabilization, particularly at the NgBR βD-βE loop, highlighting it as an allosteric hub. Functional mutagenesis scanning identified NgBRS249 as critical for enzymatic activity, independent of structural perturbations. Network analysis of MD simulations pinpointed this residue as a central node in inter-subunit communication, with perturbations disrupting downstream allosteric pathways, altering enzymatic activity. Our findings reveal a dynamic regulatory network centered at the inter-subunit interface, wherein specific NgBR residues modulate DHDDS activity through allosteric signaling. This work elucidates a conserved mechanism of subunit coordination in long-chain cis-prenyltransferases and suggests avenues for therapeutic targeting of hcis-PT-related disorders.

Prenyl chains come in multiple sizes, fulfilling unique and essential functions across all domains of life. Prenyl chains are synthesized by prenyltransferase proteins. Despite their structural similarity, prenyltransferases exhibit substantial functional diversity to create lipophilic products of varying lengths. Human cis-prenyltransferase (h-cisPT) is a tetrameric enzyme responsible for the synthesis of long prenyl chains, consisting of 20-prenyl-unit products that are essential to specific posttranslational modifications such as N-glycosylation upon downstream processing. These long products are hypothesized to transfer from h-cisPT to the ER membrane, but the mechanism of this transfer is not known. We use molecular dynamics simulations to identify a consistent membrane binding pose for h-cisPT. By quantifying protein-membrane contacts, we identify the aromatic amino acid residues in the conserved catalytic domain as critical to membrane binding. Determining relative protein-membrane binding free energies through free energy perturbation highlights the importance of these residues for membrane association, as mutations lower membrane affinity by as much as 27 kcal/mol. These results are validated using FRET to demonstrate decreased catalytic activity and membrane binding in response to mutation. Together, our results suggest a possible mechanism for prenyl substrate transfer, where key aromatic residues facilitate h-cisPT binding to the ER membrane in an orientation that holds the substrate-containing active site near the membrane surface. Molecular dynamics simulations of the mutant exhibiting lower FRET show greater orientational variability relative to wild type. This evidence for a specific orientation of h-cisPT provides a structural basis for isoprenoid association to the membrane during synthesis and prior to its release.

Protein prenylation plays a critical role in regulating the cellular localization of small GTPases and is essential for multiple myeloma (MM) pathology. Geranylgeranyl diphosphate synthase (GGPPS), producing a key prenylation moiety, exists in a dimeric or hexameric form, depending on the species. However, the functional significance of this oligomerization remains unclear. Using crystallography, mass spectrometry, and fluorescence spectroscopy, we show that the GGPPSR235C mutant-found in the widely studied MM cell line RPMI-8226-exhibits weakened inter-dimer interactions, reduced hexamer stability, and increased apparent substrate affinity and product release kinetics. These effects are even more pronounced in a dimeric mutant, GGPPSY246D, demonstrating that interdimer interactions within the hexamer help stabilize a lid region over the active site, thereby stabilizing product binding in an inhibitory conformation. Together, these findings reveal that hexamerization regulates GGPPS activity through product inhibition and underscore the importance of cell line selection and characterization in drug discovery efforts.

Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.

The human cis-prenyltransferase complex (hcis-PT) plays a major role in protein N-glycosylation by synthesizing the precursor for the glycan carrier dolichol-phosphate. Expressed in all human cells, hcis-PT is a heterotetramer consisting of two heterodimers formed between the catalytic subunit dehydrodolichyl diphosphate synthase (DHDDS) and the regulatory subunit Nogo-B receptor (NgBR). Although NgBR has no endogenous cis-prenyltransferase activity, it accelerates the activity of DHDDS ∼400-fold.

Klotho, a 1012 amino acid transmembrane protein, is a potent tumor suppressor in different cancer types. Klotho is composed of two internal repeats KL1 and KL2, and the tumor suppressor activity is primarily attributed to the KL1 domain. Despite its significant role in regulating various cancer-related pathways, the precise mechanism underlying its tumor suppressor activity remains unresolved. In this study, we aimed to identify the sequence responsible for the tumor suppressor function of Klotho and gain insights into its mechanism of action. To accomplish this, we generated expression vectors of truncated KL1 at the C and N-terminal regions and evaluated their ability to inhibit the colony formation of several cancer cell lines. Our findings demonstrated that truncated KL1 1–340 (KL340) effectively inhibited colony formation similar to KL1, while truncated KL1 1–320 (KL320) lost this activity. Furthermore, this correlated with the inhibitory effect of KL1 and KL340 on the Wnt/β-catenin pathway, whereas KL320 had no effect. Transcriptomic analysis of MCF-7 cells expressing the constructs revealed enriched pathways associated with tumor suppressor activity in KL1 and KL340. Interestingly, the α-fold predictor tool highlighted distinct differences in the α and β sheets of the TIM barrel fold of the truncated Klotho constructs, adding to our understanding of their structural variations. In summary, this study identified the 340 N-terminal amino acids as the sequence that possesses Klotho’s tumor suppressor activity and reveals a critical role in the 320–340 sequence for this function. It also provides a foundation for the development of Klotho-based therapeutic approaches for cancer treatment.

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

Voltage-gated proton channels (HV1) harbor four transmembrane helices and are devoid of a canonical ion-conductive pore, rendering them non-conventional voltage-gated cation channels. Their identification in unicellular species, such as the marine coccolithophore Emiliania huxleyi (EhHV1), has unraveled an intrinsic structural and functional diversity within this channel family. Indeed, sequence analysis of EhHV1 predicts a distinctive extracellular domain (ECD), which is absent from mammalian HV channels and enriched in histidine residues. Moreover, key histidine residues responsible for zinc-dependent regulation of human HV1 are not conserved in EhHV1. Here, we determined the contribution of the ECD of EhHV1 to zinc-mediated channel inhibition and voltage-dependent channel activity. Following detergent-mediated solubilization and purification of EhHV1 overexpressed in insect cells, size-exclusion chromatography-coupled multiangle light scattering and crosslinking analyses revealed that the purified (>99%) EhHV1 maintains a dimeric stoichiometry as their mammalian orthologs. Furthermore, the purified EhHV1 retains functionality as determined by a proton flux assay following proteoliposome reconstitution. To resolve the structural role of the ECD in EhHV1, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses. Intriguingly, incubation of EhHV1 with zinc resulted in significantly reduced deuterium uptake in the ECD, indicating its involvement in zinc binding. Notably, we also observed reduced deuterium exchange around R282 of the transmembrane S4 helix (R3), consistent with the notion that zinc binding prevents its intermittent exposure to the intracellular side of the membrane, thereby reducing channel opening probability. Moreover, voltage-clamp analyses showed that while the deletion of the ECD in EhHV1 renders the channel non-functional, mutating all ten ECD histidine residues (H163-206A) abolished zinc-mediated channel inhibition. Thus, we hypothesize that the EhHV1 ECD exhibits complex zinc-binding properties and serves as an allosteric site involved in the mobility of the S4 segment.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with Developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Prenyltransferases are a diverse category of proteins responsible for the synthesis of isoprenoids encompassing non-polar prenyl chains of varying lengths, used to synthesize a number of valuable compounds. In humans, the protein cis-prenyltransferase (cis-PT) forms an enzymatic complex that synthesizes products with long polyisoprene tails, essential for protein N-glycosylation. We have previously shown that the product is expulsed to the membrane via an outlet identified by crystallographic analysis of the enzyme along its catalytic cycle.