Kinetic and Spectroscopic Investigation of the Y157F and C93G/Y157F Variants of Cysteine Dioxygenase: Dissecting the Roles of the Second-Sphere Residues C93 and Y157.

In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (Mus musculus CDO numbering) and a nearby tyrosine at position 157 (Cys-Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys-Tyr cross-link during repeated enzyme turnover increases CDO's catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys-Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O2, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.

Engineering of the Lrp/AsnC-type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L-cysteine biosynthesis pathway in Escherichia coli.

L-cysteine is an important sulfur-containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high-throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L-cysteine-related genes one by one. To overcome this limitation, in this study, a biosensor-based approach for multilevel biosynthetic pathway optimization of L-cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecRN29Y/C81E/M90Q/M99E variant-based biosensor with improved specificity and an 8.71-fold increase in dynamic range. Using the developed biosensor, we performed high-throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29-fold increase in L-cysteine production. Molecular dynamics (MD) simulations and electrophoretic mobility shift analysis (EMSA) showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L-cysteine, which enhances transcriptional activation. Overall, our biosensor-based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Genetic variants of folate metabolism and the risk of multiple sclerosis.

BACKGROUND AND AIMS: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown cause. Alterations in one-carbon metabolism have impact in the pathophysiology by genetic susceptibility to MS and increased the risk of MS. The aim of this study was to investigate the contribution of the gene polymorphism on Methylenetetrahydrofolate Reductase (MTHFR), Methionine Synthase Reductase (MTRR), Methionine Synthase (MTR) enzymes and of the essential factors (homocysteine, Hcy; cysteine, Cys; and vitamin B12, VitB12) in folate metabolism. METHODS: Eligible MS patients (n = 147) and health controls (n = 127) were participated. The gene polymorphisms were analyzed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and the levels of plasma Hcy, Cys and VitB12 were measured by Enzyme Linked Immunuabsorbent Assay (ELISA). RESULTS AND CONCLUSION: Our results showed that the levels of Hcy and VitB12 were lower and the levels of Cys were higher in MS compared to controls. The observation of high Cys values in all 3 gene polymorphisms suggests that the transsulfiration pathway of Hcy is directed towards Cys formation since the methionine synthesis pathway does not work. We could not find any association with all gene polymorphisms with the risk of MS. The T allele of MTHFR C677T and G allele of MTR A2756G are risk factors for serum Cys level on MS. As for MTR A2756G, serum vitB12 was observed in MS patients with G allele.

A dynamic regulatory switch for phase separation of FUS protein: Zinc ions and zinc finger domain.

Zinc is an important trace element in the human body, and its homeostasis is closely related to amyotrophic lateral sclerosis (ALS). Cytoplasmic FUS proteins from patients with ALS aggregate their important pathologic markers. Liquid-liquid phase separation (LLPS) of FUS can lead to its aggregation. However, whether and how zinc homeostasis affects the aggregation of disease-associated FUS proteins in the cytoplasm remains unclear. Here, we found that zinc ion enhances LLPS and promotes the aggregation in the cytoplasm for FUS protein. In the FUS, the cysteine of the zinc finger (ZnF), recognizes and binds to zinc ions, reducing droplet mobility and enhancing protein aggregation in the cytoplasm. The mutation of FUS cysteine disrupts the dynamic regulatory switch of zinc ions and ZnF, resulting in insensitivity to zinc ions. These results suggest that the dynamic regulation of LLPS by binding with zinc ions may be a widespread mechanism and provide a new understanding of neurological diseases such as ALS and other ZnF protein-related diseases.

The mechanism of covalent inhibition of LAR phosphatase by illudalic acid.

Leukocyte antigen-related (LAR) phosphatase is a receptor-type protein tyrosine phosphatase involved in cellular signaling and associated with human disease including cancer and metabolic disorders. Selective inhibition of LAR phosphatase activity by well characterized and well validated small molecules would provide key insights into the roles of LAR phosphatase in health and disease, but identifying selective inhibitors of LAR phosphatase activity has been challenging. Recently, we described potent and selective inhibition of LAR phosphatase activity by the fungal natural product illudalic acid. Here we provide a detailed biochemical characterization of the adduct formed between LAR phosphatase and illudalic acid. A mass spectrometric analysis indicates that two cysteine residues are covalently labeled by illudalic acid and a related analog. Mutational analysis supports the hypothesis that inhibition of LAR phosphatase activity is due primarily to the adduct with the catalytic cysteine residue. A computational study suggests potential interactions between the illudalic acid moiety and the enzyme active site. Taken together, these data offer novel insights into the mechanism of inhibition of LAR phosphatase activity by illudalic acid.

Ancient Loss of Catalytic Selenocysteine Spurred Convergent Adaptation in a Mammalian Oxidoreductase.

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration.

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.

Cysteine- and glycine-rich protein 1 predicts prognosis and therapy response in patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis. The current risk stratification system is essential but remains insufficient to select the best schedules. Cysteine-rich protein 1 (CSRP1) is a member of the CSRP family and associated with poor clinicopathological features in many tumors. This study aimed to explore the clinical significance and molecular mechanisms of cysteine- and glycine-rich protein 1 (CSRP1) in AML. RT-qPCR was used to detect the relative expression of CSRP1 in our clinical cohort. Functional enrichment analysis of CSRP1-related differentially expressed genes was carried out by GO/KEGG enrichment analysis, immune cell infiltration analysis, and protein-protein interaction (PPI) network. The OncoPredict algorithm was implemented to explore correlations between CSRP1 and drug resistance. CSRP1 was highly expressed in AML compared with normal samples. High CSRP1 expression was an independent poor prognostic factor. Functional enrichment analysis showed neutrophil activation and apoptosis were associated with CSRP1. In the PPI network, 19 genes were present in the most significant module, and 9 of them were correlated with AML prognosis. The high CSRP1 patients showed higher sensitivity to 5-fluorouracil, gemcitabine, rapamycin, cisplatin and lower sensitivity to fludarabine. CSRP1 may serve as a potential prognostic marker and a therapeutic target for AML in the future.

Evolutionary Analysis of Cnidaria Small Cysteine-Rich Proteins (SCRiPs), an Enigmatic Neurotoxin Family from Stony Corals and Sea Anemones (Anthozoa: Hexacorallia).

Cnidarians (corals, sea anemones, and jellyfish) produce toxins that play central roles in key ecological processes, including predation, defense, and competition, being the oldest extant venomous animal lineage. Cnidaria small cysteine-rich proteins (SCRiPs) were the first family of neurotoxins detected in stony corals, one of the ocean's most crucial foundation species. Yet, their molecular evolution remains poorly understood. Moreover, the lack of a clear classification system has hindered the establishment of an accurate and phylogenetically informed nomenclature. In this study, we extensively surveyed 117 genomes and 103 transcriptomes of cnidarians to identify orthologous SCRiP gene sequences. We annotated a total of 168 novel putative SCRiPs from over 36 species of stony corals and 12 species of sea anemones. Phylogenetic reconstruction identified four distinct SCRiP subfamilies, according to strict discrimination criteria based on well-supported monophyly with a high percentage of nucleotide and amino acids' identity. Although there is a high prevalence of purifying selection for most SCRiP subfamilies, with few positively selected sites detected, a subset of Acroporidae sequences is influenced by diversifying positive selection, suggesting potential neofunctionalizations related to the fine-tuning of toxin potency. We propose a new nomenclature classification system relying on the phylogenetic distribution and evolution of SCRiPs across Anthozoa, which will further assist future proteomic and functional research efforts.

Mechanisms and functions of SUMOylation in health and disease: a review focusing on immune cells.

SUMOylation, which is a type of post-translational modification that involves covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to target substrates, regulates various important molecular and cellular processes, including transcription, the cell cycle, cell signaling, and DNA synthesis and repair. Newly synthesized SUMO is immature and cleaved by the SUMO-specific protease family, resulting in exposure of the C-terminal Gly-Gly motif to become the mature form. In the presence of ATP, mature SUMO is conjugated with the activating enzyme E1 through the cysteine residue of E1, followed by transfer to the cysteine residue of E2-conjugating enzyme Ubc9 in humans that recognizes and modifies the lysine residue of a substrate protein. E3 SUMO ligases promote SUMOylation. SUMOylation is a reversible modification and mediated by SUMO-specific proteases. Cumulative studies have indicated that SUMOylation affects the functions of protein substrates in various manners, including cellular localization and protein stability. Gene knockout studies in mice have revealed that several SUMO cycling machinery proteins are crucial for the development and differentiation of various cell lineages, including immune cells. Aberrant SUMOylation has been implicated in several types of diseases, including cancers, cardiovascular diseases, and autoimmune diseases. This review summarizes the biochemistry of SUMO modification and the general biological functions of proteins involved in SUMOylation. In particular, this review focuses on the molecular mechanisms by which SUMOylation regulates the development, maturation, and functions of immune cells, including T, B, dendritic, and myeloid cells. This review also discusses the underlying relevance of disruption of SUMO cycling and site-specific interruption of SUMOylation on target proteins in immune cells in diseases, including cancers and infectious diseases.

Altered Protein Dynamics and a More Reactive Catalytic Cysteine in a Neurodegeneration-associated UCHL1 Mutant.

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.

Cysteine and glycine-rich protein 2 retards platelet-derived growth factor-BB-evoked phenotypic transition of airway smooth muscle cells by decreasing YAP/TAZ activity.

Cysteine and glycine-rich protein 2 (Csrp2) has emerged as a key factor in controlling the phenotypic modulation of smooth muscle cells. The phenotypic transition of airway smooth muscle cells (ASMCs) is a pivotal step in developing airway remodeling during the onset of asthma. However, whether Csrp2 mediates the phenotypic transition of ASMCs in airway remodeling during asthma onset is undetermined. This work aimed to address the link between Csrp2 and the phenotypic transition of ASMCs evoked by platelet-derived growth factor (PDGF)-BB in vitro. The overexpression or silencing of Csrp2 in ASMCs was achieved through adenovirus-mediated gene transfer. The expression of mRNA was measured by quantitative real-time-PCR. Protein levels were determined through Western blot analysis. Cell proliferation was detected by EdU assay and Calcein AM assays. Cell cycle distribution was assessed via fluorescence-activated cell sorting assay. Cell migration was evaluated using the scratch-wound assay. The transcriptional activity of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) was measured using the luciferase reporter assay. A decline in Csrp2 level occurred in PDGF-BB-stimulated ASMCs. Increasing Csrp2 expression repressed the PDGF-BB-evoked proliferation and migration of ASMCs. Moreover, increasing Csrp2 expression impeded the phenotypic change of PDGF-BB-stimulated ASMCs from a contractile phenotype into a synthetic/proliferative phenotype. On the contrary, the opposite effects were observed in Csrp2-silenced ASMCs. The activity of YAP/TAZ was elevated in PDGF-BB-stimulated ASMCs, which was weakened by Csrp2 overexpression or enhanced by Csrp2 silencing. The YAP/TAZ activator could reverse Csrp2-overexpression-mediated suppression of the PDGF-BB-evoked phenotypic switching of ASMCs, while the YAP/TAZ suppressor could dimmish Csrp2-silencing-mediated enhancement on PDGF-BB-evoked phenotypic switching of ASMCs. In summary, Csrp2 serves as a determinant for the phenotypic switching of ASMCs. Increasing Csrp2 is able to impede PDGF-BB-evoked phenotypic change of ASMCs from a synthetic phenotype into a synthetic/proliferative phenotype through the effects on YAP/TAZ. This work implies that Csrp2 may be a key player in airway remodeling during the onset of asthma.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Channel Gating in Kalium Channelrhodopsin Slow Mutants.

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K+ than to Na+ and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the transmembrane helix 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate. The phenotype of the corresponding mutants in channelrhodopsin 2 is attributed to breaking of a specific interhelical hydrogen bond (the "DC gate"). Unlike CrChR2 and other ChRs with long distance "DC gates", the HcKCR1 structure does not reveal any hydrogen bonding partners to Cys110, indicating that the mutant phenotype is likely caused by disruption of direct interaction between this residue and the chromophore. In HcKCR1_C110A, fast photochemical conversions corresponding to channel gating were followed by dramatically slower absorption changes. Full recovery of the unphotolyzed state in HcKCR1_C110A was extremely slow with two time constants 5.2 and 70 min. Analysis of the light-minus-dark difference spectra during these slow processes revealed accumulation of at least four spectrally distinct blue light-absorbing photocycle intermediates, L, M1 and M2, and a UV light-absorbing form, typical of bacteriorhodopsin-like channelrhodopsins from cryptophytes. Our results contribute to better understanding of the mechanistic links between the chromophore photochemistry and channel conductance, and provide the basis for using HcKCR1_C110A as an optogenetic tool.

Re-evaluation of a Fibrillin-1 Gene Variant of Uncertain Significance Using the ClinGen Guidelines.

Background: Marfan syndrome (MFS) is caused by fibrillin-1 gene (FBN1) variants. Mutational hotspots and/or well-established critical functional domains of FBN1 include cysteine residues, calcium-binding consensus sequences, and amino acids related to interdomain packaging. Previous guidelines for variant interpretation do not reflect the features of genes or related diseases. Using the Clinical Genome Resource (ClinGen) FBN1 variant curation expert panel (VCEP), we re-evaluated FBN1 germline variants reported as variants of uncertain significance (VUSs). Methods: We re-evaluated 26 VUSs in FBN1 reported in 161 patients with MFS. We checked the variants in the Human Genome Mutation Database, ClinVar, and VarSome databases and assessed their allele frequencies using the gnomAD database. Patients' clinical information was reviewed. Results: Four missense variants affecting cysteines (c.460T>C, c.1006T>C, c.5330G>C, and c.8020T>C) were reclassified as likely pathogenic and were assigned PM1_strong or PM1. Two intronic variants were reclassified as benign by granting BA1 (stand-alone). Four missense variants were reclassified as likely benign. BP5 criteria were applied in cases with an alternate molecular basis for disease, one of which (c.7231G>A) was discovered alongside a pathogenic de novo COL3A1 variant (c.1988G>T, p.Gly633Val). Conclusions: Considering the high penetrance of FBN1 variants and clinical variability of MFS, the detection of pathogenic variants is important. The ClinGen FBN1 VCEP encompasses mutational hotspots and/or well-established critical functional domains and adjusts the criteria specifically for MFS; therefore, it is beneficial not only for identifying pathogenic FBN1 variants but also for distinguishing these variants from those that cause other connective tissue disorders with overlapping clinical features.

Acetylenic tricyclic bis-(cyano enone) interacts with Cys 374 of actin, a residue necessary for stress fiber formation and cell migration.

The migratory and invasive potential of tumour cells relies on the actin cytoskeleton. We previously demonstrated that the tricyclic compound, TBE-31, inhibits actin polymerization and here we further examine the precise interaction between TBE-31 and actin. We demonstrate that iodoacetamide, a cysteine (Cys) alkylating agent, interferes with the ability of TBE-31 to interact with actin. In addition, in silico analysis identified Cys 217, Cys 272, Cys 285 and Cys 374 as potential binding sites for TBE-31. Using mass spectrometry analysis, we determined that TBE-31 associates with actin with a stoichiometric ratio of 1:1. We mutated the identified cysteines of actin to alanine and performed a pull-down analysis with a biotin labeled TBE-31 and demonstrated that by mutating Cys 374 to alanine the association between TBE-31 and actin was significantly reduced, suggesting that TBE-31 binds to Cys 374. A characterization of the NIH3T3 cells overexpressing eGFP-actin-C374A showed reduced stress fiber formation, suggesting Cys 374 is necessary for efficient incorporation into filamentous actin. Furthermore, migration of eGFP-Actin-WT expressing cells were observed to be inhibited by TBE-31, however fewer eGFP-Actin-C374A expressing cells were observed to migrate compared to the cells expressing eGFP-Actin-WT in the presence or absence of TBE-31. Taken together, our results suggest that TBE-31 binds to Cys 374 of actin to inhibit actin stress fiber formation and may potentially be a mechanism through which TBE-31 inhibits cell migration.

Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the promyelocytic leukemia-RING-B-box-coiled-coil (PML-RBCC) domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F, and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P, and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (Tm) reduction of 30 °C compared with the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. The spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F, and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F, and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213, and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.