Sci Total Environ. 2023 Mar 1:162517. doi: 10.1016/j.scitotenv.2023.162517. Online ahead of print.

ABSTRACT

The continuous worldwide seagrasses decline calls for immediate actions in order to preserve this precious marine ecosystem. The main stressors that have been linked with decline in seagrasses are 1) the increasing ocean temperature due to climate change and 2) the continuous inputs of nutrients (eutrophication) associated with coastal human activities. To avoid the loss of seagrass populations, an "early warning" system is needed. We used Weighed Gene Co-expression Network Analysis (WGCNA), a systems biology approach, to identify potential candidate genes that can provide an early warning signal of stress in the Mediterranean iconic seagrass Posidonia oceanica, anticipating plant mortality. Plants were collected from both eutrophic (EU) and oligotrophic (OL) environments and were exposed to thermal and nutrient stress in a dedicated mesocosm. By correlating the whole-genome gene expression after 2-weeks exposure with the shoot survival percentage after 5-weeks exposure to stressors, we were able to identify several transcripts that indicated an early activation of several biological processes (BP) including: protein metabolic process, RNA metabolic process, organonitrogen compound biosynthetic process, catabolic process and response to stimulus, which were shared among OL and EU plants and among leaf and shoot apical meristem (SAM), in response to excessive heat and nutrients. Our results suggest a more dynamic and specific response of the SAM compared to the leaf, especially the SAM from plants coming from a more stressful environment appeared more dynamic than the SAM from a more pristine environment. A vast list of potential molecular markers is also provided that can be used as targets to assess field samples.

PMID:36868282 | DOI:10.1016/j.scitotenv.2023.162517

Cell. 2023 Mar 1:S0092-8674(23)00106-X. doi: 10.1016/j.cell.2023.02.004. Online ahead of print.

ABSTRACT

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.

PMID:36868219 | DOI:10.1016/j.cell.2023.02.004

Horm Behav. 2023 Mar 1;151:105338. doi: 10.1016/j.yhbeh.2023.105338. Online ahead of print.

ABSTRACT

Although copulatory behavior is thought to have a strong innate basis in mice, there is also clear evidence that sexual experience shapes its expression. Reinforcement of behavior through rewarding genital tactile stimulation is a primary candidate mechanism for this modification. In rats, manual tactile clitoral stimulation is rewarding only when it is temporally distributed, which is hypothesized to result from an innate preference for species-typical copulatory patterning. Here we test this hypothesis using mice, which have a temporal copulatory pattern which is distinctly less temporally distributed than that of rats. Female mice received manual clitoral stimulation which was either temporally continuous every second, or stimulation which was temporally distributed, occurring every 5 s, This pattern of stimulation was paired with environmental cues in a conditioned place preference apparatus to assess reward. Neural activation in response to this stimulation was evaluated by measuring FOS immunoreactivity. Results indicated that both temporal patterns of clitoral stimulation were rewarding, but that continuous stimulation better reproduced brain activation associated with sexual reward. Furthermore, continuous, but not distributed stimulation elicited a lordosis response in some females, and this response increased within and across days. Sexual reward, neural activation and lordosis resulting from tactile genital stimulation were eliminated by ovariectomy and restored with combined 17β-estradiol and progesterone treatment but not 17β-estradiol treatment alone. These observations are consistent with the hypothesis that sexual reward resulting from species-typical genital tactile stimulation has a permissive effect on copulatory behavior of female mice.

PMID:36868148 | DOI:10.1016/j.yhbeh.2023.105338

Biosens Bioelectron. 2023 Feb 24;227:115178. doi: 10.1016/j.bios.2023.115178. Online ahead of print.

ABSTRACT

Seasonal outbreaks of respiratory viral infections remain a global concern, with increasing morbidity and mortality rates recorded annually. Timely and false responses contribute to the widespread of respiratory pathogenic diseases owing to similar symptoms at an early stage and subclinical infection. The prevention of emerging novel viruses and variants is also a big challenge. Reliable point-of-care diagnostic assays for early infection diagnosis play a critical role in the response to threats of epidemics or pandemics. We developed a facile method for specifically identifying different viruses based on surface-enhanced Raman spectroscopy (SERS) with pathogen-mediated composite materials on Au nanodimple electrodes and machine learning (ML) analyses. Virus particles were trapped in three-dimensional plasmonic concave spaces of the electrode via electrokinetic preconcentration, and Au films were simultaneously electrodeposited, leading to the acquisition of intense and in-situ SERS signals from the Au-virus composites for ultrasensitive SERS detection. The method was useful for rapid detection analysis (<15 min), and the ML analysis for specific identification of eight virus species, including human influenza A viruses (i.e., H1N1 and H3N2 strains), human rhinovirus, and human coronavirus, was conducted. The highly accurate classification was achieved using the principal component analysis-support vector machine (98.9%) and convolutional neural network (93.5%) models. This ML-associated SERS technique demonstrated high feasibility for direct multiplex detection of different virus species for on-site applications.

PMID:36867960 | DOI:10.1016/j.bios.2023.115178

Chem Res Toxicol. 2023 Mar 3. doi: 10.1021/acs.chemrestox.2c00390. Online ahead of print.

ABSTRACT

Little is known about the chemical exposures that electronic cigarette (EC) users receive and emit during JUUL vaping and if exposures produce symptoms dose dependently. This study examined chemical exposure (dose), retention, symptoms during vaping, and the environmental accumulation of exhaled propylene glycol (PG), glycerol (G), nicotine, and menthol in a cohort of human participants who vaped JUUL "Menthol" ECs. We refer to this environmental accumulation as "EC exhaled aerosol residue" (ECEAR). Chemicals were quantified using gas chromatography/mass spectrometry in JUUL pods before and after use, lab-generated aerosols, human exhaled aerosols, and in ECEAR. Unvaped JUUL "Menthol" pods contained ∼621.3 mg/mL of G, ∼264.9 mg/mL of PG, ∼59.3 mg/mL of nicotine, ∼13.3 mg/mL of menthol, and ∼0.1 mg/mL of the coolant WS-23. Eleven experienced male EC users (aged 21-26) provided exhaled aerosol and residue samples before and after vaping JUUL pods. Participants vaped ad libitum for 20 min, while their average puff count (22 ± 6.4) and puff duration (4.4 ± 2.0) were recorded. The transfer efficiency of nicotine, menthol, and WS-23 from the pod fluid into the aerosol varied with each chemical and was generally similar across flow rates (9-47 mL/s). At 21 mL/s, the average mass of each chemical retained by the participants who vaped 20 min was 53.2 ± 40.3 mg for G, 18.9 ± 14.3 mg for PG, 3.3 ± 2.7 mg for nicotine, and 0.5 ± 0.4 mg for menthol, with retention deduced to be ∼90-100% for each chemical. There was a significant positive relationship between the number of symptoms during vaping and total chemical mass retained. ECEAR accumulated on enclosed surfaces where it could contribute to passive exposure. These data will be valuable to researchers studying human exposure to EC aerosols and agencies that regulate EC products.

PMID:36867872 | DOI:10.1021/acs.chemrestox.2c00390

Sci Adv. 2023 Mar 3;9(9):eabp8314. doi: 10.1126/sciadv.abp8314. Epub 2023 Mar 3.

ABSTRACT

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

PMID:36867694 | DOI:10.1126/sciadv.abp8314

J Med Virol. 2023 Mar 3. doi: 10.1002/jmv.28632. Online ahead of print.

ABSTRACT

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne emerging infectious disease with an increasingly global concern. Sex difference in infectious diseases is an important public health problem. A comparative study on sex differences in SFTS incidence and fatality was conducted using all laboratory-confirmed cases in mainland China during 2010-2018. Females had significantly higher average annual incidence rate (AAIR) with a RR of 1.17 (95% CI 1.11-1.22; p˂0.0001), but significantly lower-case fatality rate (CFR) with an OR of 0.73 (95% CI 0.61-0.87; p=0.001). The significant differences in AAIR and CFR were observed in age groups of 40-69 and 60-69 years, respectively (both p<0.05). There was a rising incidence and declining CFR along with epidemic years. After adjusting for age, temporal and spatial distribution, agricultural setting and onset-to-diagnosis interval, the female-to-male difference in either AAIR or CFR remained significant. The underlying biological mechanisms of the sex-based differences that the females are more prone to get the disease, but less likely for a fatal outcome deserve further investigations. This article is protected by copyright. All rights reserved.

PMID:36866702 | DOI:10.1002/jmv.28632

Circ Genom Precis Med. 2023 Mar 3:e003900. doi: 10.1161/CIRCGEN.122.003900. Online ahead of print.

NO ABSTRACT

PMID:36866680 | DOI:10.1161/CIRCGEN.122.003900

Essays Biochem. 2023 Mar 3:EBC20220177. doi: 10.1042/EBC20220177. Online ahead of print.

ABSTRACT

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein-RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid-liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

PMID:36866608 | DOI:10.1042/EBC20220177

Biotechnol J. 2023 Mar 2:e2200622. doi: 10.1002/biot.202200622. Online ahead of print.

ABSTRACT

Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions towards a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2 O2 without the addition of NAD(P)H. In this work protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2 O2 . The full-length enzyme (CYP116B5-fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2 O2 (CYP116B5-hd) and sarcosine (CYP116B5-SOX) as source of electrons. CYP116B5-SOX performs better than CYP116B5-fl and CYP116B5-hd showing 10 folds and 3 folds higher activity, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class. This article is protected by copyright. All rights reserved.

PMID:36866427 | DOI:10.1002/biot.202200622

Front Pharmacol. 2023 Feb 14;14:1122388. doi: 10.3389/fphar.2023.1122388. eCollection 2023.

ABSTRACT

Cancer is the third leading cause of premature death in sub-Saharan Africa. Cervical cancer has the highest number of incidences in sub-Saharan Africa due to high HIV prevalence (70% of global cases) in African countries which is linked to increasing the risk of developing cervical cancer, and the continuous high risk of being infected with Human papillomavirus In 2020, the risk of dying from cancer amongst women was higher in Eastern Africa (11%) than it was in Northern America (7.4%). Plants continue to provide unlimited pharmacological bioactive compounds that are used to manage various illnesses, including cancer. By reviewing the literature, we provide an inventory of African plants with reported anticancer activity and evidence supporting their use in cancer management. In this review, we report 23 plants that have been used for cancer management in Africa, where the anticancer extracts are usually prepared from barks, fruits, leaves, roots, and stems of these plants. Extensive information is reported about the bioactive compounds present in these plants as well as their potential activities against various forms of cancer. However, information on the anticancer properties of other African medicinal plants is insufficient. Therefore, there is a need to isolate and evaluate the anticancer potential of bioactive compounds from other African medicinal plants. Further studies on these plants will allow the elucidation of their anticancer mechanisms of action and allow the identification of phytochemicals that are responsible for their anticancer properties. Overall, this review provides consolidated and extensive information not only on diverse medicinal plants of Africa but on the different types of cancer that these plants are used to manage and the diverse mechanisms and pathways that are involved during cancer alleviation.

PMID:36865913 | PMC:PMC9971233 | DOI:10.3389/fphar.2023.1122388

Biochem Biophys Rep. 2023 Feb 16;34:101438. doi: 10.1016/j.bbrep.2023.101438. eCollection 2023 Jul.

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death among non-contagious diseases in the world. PDE inhibitors are among current medicines prescribed for COPD treatment of which, PDE-4 family is the predominant PDE isoform involved in hydrolyzing cyclic adenosine monophosphate (cAMP) that regulates the inflammatory responses in neutrophils, lymphocytes, macrophages and epithelial cells The aim of this study is to investigate the cellular and molecular mechanisms of cAMP-PDE signaling, as an important pathway in the treatment management of patients with COPD. In this review, a comprehensive literature review was performed about the effect of PDEs in COPD. Generally, PDEs are overexpressed in COPD patients, resulting in cAMP inactivation and decreased cAMP hydrolysis from AMP. At normal amounts, cAMP is one of the essential agents in regulating metabolism and suppressing inflammatory responses. Low amount of cAMP lead to activation of downstream inflammatory signaling pathways. PDE4 and PDE7 mRNA transcript levels were not altered in polymorphonuclear leukocytes and CD8 lymphocytes originating from the peripheral venous blood of stable COPD subjects compared to healthy controls. Therefore, cAMP-PDE signaling pathway is one of the most important signaling pathways involved in COPD. By examining the effects of different drugs in this signaling pathway critical steps can be taken in the treatment of this disease.

PMID:36865738 | PMC:PMC9971187 | DOI:10.1016/j.bbrep.2023.101438

MAbs. 2023 Jan-Dec;15(1):2183540. doi: 10.1080/19420862.2023.2183540.

ABSTRACT

Bispecific antibodies are molecules with versatile modes of action and applications for therapy. They are commonly developed as T-cell engagers (TCE), which simultaneously target an antigen expressed by tumor cells and CD3 expressed by T-cells, thereby inducing T-cell-mediated target cell killing. There is growing evidence that the molecular composition and valency for the target antigen influence the activity of TCEs. Here, the eIg platform technology was used to generate a set of bispecific TCEs targeting epidermal growth factor receptors (EGFR) and CD3. These molecules either included or lacked an Fc region and exhibited one binding site for CD3 and either one or two binding sites for EGFR (1 + 1 or 2 + 1 formats) utilizing different molecular arrangements of the binding sites. In total, 11 different TCE formats were analyzed for binding to target cells and T cells, T cell-mediated killing of tumor cells, and for the activation of T cells (release of cytokines and proliferation of T-cells). Bivalent binding to EGFR strongly increased binding and T cell-mediated killing. However, the molecular composition and position of the CD3-binding arm also affected target cell killing, cytokine release, and T-cell proliferation. Our findings support that screening of a panel of formats is beneficial to identify the most potent bispecific TCE, and that format matters.

PMID:36864566 | DOI:10.1080/19420862.2023.2183540

J Transl Med. 2023 Mar 2;21(1):163. doi: 10.1186/s12967-023-04010-z.

ABSTRACT

BACKGROUND: Gastric cancer (GC) is a major cancer burden throughout the world with a high mortality rate. The performance of current predictive and prognostic factors is still limited. Integrated analysis is required for accurate cancer progression predictive biomarker and prognostic biomarkers that help to guide therapy.

METHODS: An AI-assisted bioinformatics method that combines transcriptomic data and microRNA regulations were used to identify a key miRNA-mediated network module in GC progression. To reveal the module's function, we performed the gene expression analysis in 20 clinical samples by qRT-PCR, prognosis analysis by multi-variable Cox regression model, progression prediction by support vector machine, and in vitro studies to elaborate the roles in GC cells migration and invasion.

RESULTS: A robust microRNA regulated network module was identified to characterize GC progression, which consisted of seven miR-200/183 family members, five mRNAs and two long non-coding RNAs H19 and CLLU1. Their expression patterns and expression correlation patterns were consistent in public dataset and our cohort. Our findings suggest a two-fold biological potential of the module: GC patients with high-risk score exhibited a poor prognosis (p-value < 0.05) and the model achieved AUCs of 0.90 to predict GC progression in our cohort. In vitro cellular analyses shown that the module could influence the invasion and migration of GC cells.

CONCLUSIONS: Our strategy which combines AI-assisted bioinformatics method with experimental and clinical validation suggested that the miR-200/183 family-mediated network module as a "pluripotent module", which could be potential marker for GC progression.

PMID:36864416 | DOI:10.1186/s12967-023-04010-z

Nat Methods. 2023 Mar 2. doi: 10.1038/s41592-023-01785-3. Online ahead of print.

ABSTRACT

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

PMID:36864200 | DOI:10.1038/s41592-023-01785-3

Nat Chem Biol. 2023 Mar 2. doi: 10.1038/s41589-023-01270-0. Online ahead of print.

ABSTRACT

Much of the human proteome is involved in mRNA homeostasis, but most RNA-binding proteins lack chemical probes. Here we identify electrophilic small molecules that rapidly and stereoselectively decrease the expression of transcripts encoding the androgen receptor and its splice variants in prostate cancer cells. We show by chemical proteomics that the compounds engage C145 of the RNA-binding protein NONO. Broader profiling revealed that covalent NONO ligands suppress an array of cancer-relevant genes and impair cancer cell proliferation. Surprisingly, these effects were not observed in cells genetically disrupted for NONO, which were instead resistant to NONO ligands. Reintroduction of wild-type NONO, but not a C145S mutant, restored ligand sensitivity in NONO-disrupted cells. The ligands promoted NONO accumulation in nuclear foci and stabilized NONO-RNA interactions, supporting a trapping mechanism that may prevent compensatory action of paralog proteins PSPC1 and SFPQ. These findings show that NONO can be co-opted by covalent small molecules to suppress protumorigenic transcriptional networks.

PMID:36864190 | DOI:10.1038/s41589-023-01270-0

Nat Struct Mol Biol. 2023 Mar 2. doi: 10.1038/s41594-023-00928-6. Online ahead of print.

ABSTRACT

Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork.

PMID:36864174 | DOI:10.1038/s41594-023-00928-6

J Math Biol. 2023 Mar 3;86(4):50. doi: 10.1007/s00285-023-01877-w.

ABSTRACT

Density dependence is important in the ecology and evolution of microbial and cancer cells. Typically, we can only measure net growth rates, but the underlying density-dependent mechanisms that give rise to the observed dynamics can manifest in birth processes, death processes, or both. Therefore, we utilize the mean and variance of cell number fluctuations to separately identify birth and death rates from time series that follow stochastic birth-death processes with logistic growth. Our nonparametric method provides a novel perspective on stochastic parameter identifiability, which we validate by analyzing the accuracy in terms of the discretization bin size. We apply our method to the scenario where a homogeneous cell population goes through three stages: (1) grows naturally to its carrying capacity, (2) is treated with a drug that reduces its carrying capacity, and (3) overcomes the drug effect to restore its original carrying capacity. In each stage, we disambiguate whether the dynamics occur through the birth process, death process, or some combination of the two, which contributes to understanding drug resistance mechanisms. In the case of limited sample sizes, we provide an alternative method based on maximum likelihood and solve a constrained nonlinear optimization problem to identify the most likely density dependence parameter for a given cell number time series. Our methods can be applied to other biological systems at different scales to disambiguate density-dependent mechanisms underlying the same net growth rate.

PMID:36864131 | DOI:10.1007/s00285-023-01877-w

Nat Genet. 2023 Mar 2. doi: 10.1038/s41588-023-01329-7. Online ahead of print.

ABSTRACT

Epigenetic reprogramming in the germline contributes to the erasure of epigenetic inheritance across generations in mammals but remains poorly characterized in plants. Here we profiled histone modifications throughout Arabidopsis male germline development. We find that the sperm cell has widespread apparent chromatin bivalency, which is established by the acquisition of H3K27me3 or H3K4me3 at pre-existing H3K4me3 or H3K27me3 regions, respectively. These bivalent domains are associated with a distinct transcriptional status. Somatic H3K27me3 is generally reduced in sperm, while dramatic loss of H3K27me3 is observed at only ~700 developmental genes. The incorporation of the histone variant H3.10 facilitates the establishment of sperm chromatin identity without a strong impact on resetting of somatic H3K27me3. Vegetative nuclei harbor thousands of specific H3K27me3 domains at repressed genes, while pollination-related genes are highly expressed and marked by gene body H3K4me3. Our work highlights putative chromatin bivalency and restricted resetting of H3K27me3 at developmental regulators as key features in plant pluripotent sperm.

PMID:36864100 | DOI:10.1038/s41588-023-01329-7

Nat Commun. 2023 Mar 2;14(1):1002. doi: 10.1038/s41467-023-36597-8.

ABSTRACT

In two-component systems, the information gathered by histidine kinases (HKs) are relayed to cognate response regulators (RRs). Thereby, the phosphoryl group of the auto-phosphorylated HK is transferred to the receiver (Rec) domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec (Recinter) domain that is typically part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about discriminating features of Recinter domains. Here we study the Recinter domain of the hybrid HK CckA by X-ray crystallography and NMR spectroscopy. Strikingly, all active site residues of the canonical Rec-fold are pre-arranged for phosphoryl-binding and BeF3- binding does not alter secondary or quaternary structure, indicating the absence of allosteric changes, the hallmark of RRs. Based on sequence-covariation and modeling, we analyze the intra-molecular DHp/Rec association in hybrid HKs.

PMID:36864019 | DOI:10.1038/s41467-023-36597-8