CACHE Domain Research Articles

Amine-recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor

Bacteria possess various receptors that sense different signals and transmit information to enable an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Signals recognized by sensor domains are poorly reflected in overall sequence identity, and therefore, the identification of signals from the amino acid sequence of the sensor alone presents a challenge. Biogenic amines are of great physiological importance for microorganisms and humans. They serve as substrates for aerobic and anaerobic growth and play a role of neurotransmitters and osmoprotectants. Here, we report the identification of a sequence motif that is specific for amine-sensing sensor domains that belong to the Cache superfamily of the most abundant extracellular sensors in prokaryotes. We identified approximately 13,000 sensor histidine kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases from 8,000 bacterial and archaeal species that contain the amine-recognizing motif. The screening of compound libraries and microcalorimetric titrations of selected sensor domains confirmed their ability to specifically bind biogenic amines. Mutants in the amine-binding motif or domains that contain a single mismatch in the binding motif had either no or a largely reduced affinity for amines. We demonstrate that the amine-recognizing domain originated from the universal amino acid-sensing Cache domain, thus providing insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and therefore holds a strong promise to enable the identification of signals stimulating numerous receptors.

Structure of the photoreceptor synaptic assembly of the extracellular matrix protein pikachurin with the orphan receptor GPR179.

Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.

498 Structural Determination of the CqsR CACHE Domain and its Autoinducer

OBJECTIVES/GOALS: Our goal is to determine the structure of the CACHE domain of the Vibrio cholerae quorum sensing receptor CqsR as well as its autoinducer (AI). We are performing X-ray crystallography on the protein in its apo form, with the fractions containing the AI, and with known ligand ethanolamine (ETA). METHODS/STUDY POPULATION: We have transformed BL21(DE3) E. coli cells with a pTB146 vector to contain the gene for the CqsR CACHE domain. We grow these cells to high optical density and induce protein expression, at which point we harvest them and purify the protein. This entails lysing the cells, separating the protein with Ni-NTA resin, cleaving our protein tag, and column chromatography. With purified protein, high-throughput screens are set up to find crystallization conditions of apo CqsR, CqsR-ETA, and CqsR-AI. We then determine conditions that best lead to crystal formation and optimize them. Crystals are then diffracted with X-rays, process the data, and determine the structure of protein and AI. RESULTS/ANTICIPATED RESULTS: We anticipate finding the structure of the CqsR CACHE domain to a high resolution in addition to the identity of its autoinducer. Previous results found that the structure is homologous to another V. cholerae chemoreceptor, Mlp37, and we expect the results from this project to confirm this. In addition, we know that the autoinducer weighs approximately 62 daltons, the same as the known ligand, ethanolamine. Given that CACHE domains bind specifically to their ligands, we anticipate that the autoinducer will be structurally similar to ethanolamine. DISCUSSION/SIGNIFICANCE: The results will reveal the structure of the CqsR CACHE domain and its autoinducer. This knowledge will better allow researchers to treat cholera, as both autoinducer identity and receptor conformational changes will be uncovered, allowing for drug development to inhibit cell growth.

Orphan receptor GPR158 serves as a metabotropic glycine receptor: mGlyR.

Glycine is a major neurotransmitter involved in several fundamental neuronal processes. The identity of the metabotropic receptor mediating slow neuromodulatory effects of glycine is unknown. We identified an orphan G protein-coupled receptor, GPR158, as a metabotropic glycine receptor (mGlyR). Glycine and a related modulator, taurine, directly bind to a Cache domain of GPR158, and this event inhibits the activity of the intracellular signaling complex regulator of G protein signaling 7-G protein β5 (RGS7-Gβ5), which is associated with the receptor. Glycine signals through mGlyR to inhibit production of the second messenger adenosine 3',5'-monophosphate. We further show that glycine, but not taurine, acts through mGlyR to regulate neuronal excitability in cortical neurons. These results identify a major neuromodulatory system involved in mediating metabotropic effects of glycine, with implications for understanding cognition and affective states.

Searching for Osmosensing Determinants in Poplar Histidine-Aspartate Kinases

Previous works have shown the existence of protein partnership, belonging to a MultiStep Phosphorelay (MSP), potentially involved in osmosensing in Populus. The first actor of this signalling pathway belongs to the histidine-aspartate kinase (HK) family, which also includes the yeast osmosensor Sln1, as well as the Arabidopsis putative osmosensor AHK1. In poplar, the homologous AHK1 protein corresponds to a pair of paralogous proteins, HK1a and HK1b, exhibiting an extracellular domain (ECD), as in Sln1 and AHK1. An ECD alignment of AHK1-like proteins, from different plant species, showed a particularly well conserved ECD and revealed the presence of a cache domain. This level of conservation suggested a functional role of this domain in osmosensing. Thus, we tested this possibility by modelling assisted mutational analysis of the cache domain of the Populus HK1 proteins. The mutants were assessed for their ability to respond to different osmotic stress and the results point to an involvement of this domain in HK1 functionality. Furthermore, since HK1b was shown to respond better to stress than HK1a, these two receptors constituted a good system to search for osmosensing determinants responsible for this difference in efficiency. With domain swapping experiments, we finally demonstrated that the cache domain, as well as the second transmembrane domain, are involved in the osmosensing efficiency of these receptors.

Structural basis for environmental sensing in Pseudomonas fluorescens

Over the past decades, it has become clear that bacteria often prefer a sessile over a free‐swimming lifestyle. The transition from a mobile to an immobile, social lifestyle, called a biofilm, is induced in response to a variety of environmental stimuli, such as carbon or phosphate sources, linked to signaling via the second messenger cyclic‐di‐GMP (c‐di‐GMP). In gammaproteobacteria, c‐di‐GMP regulates cell adhesion to biotic and abiotic surfaces through the transmembrane Lap system that controls the surface presentation of large adhesin proteins. Signaling specificity is achieved through direct physical interaction between a c‐di‐GMP‐producing diguanylate cyclases (DGCs) and LapD, the c‐di‐GMP receptor central to the Lap system. To date, ten DGCs have been identified as regulators of LapD, affecting biofilm formation. However, how they differ functionally and the stimuli to which they respond are often not well understood. To shed light on the environmental sensing mechanism for DGC activation, we have determined the structure of the periplasmic domain of two DGCs in P. fluorescens, a model organism for biofilm research. These domains, belonging to the CACHE family of protein domains, protrude into the periplasm where they encounter their respective ligands, which correlates with their activation. By comparing the structures of CACHE domains and their ligand binding profiles, we identify molecular signatures that contribute to c‐di‐GMP signaling specificity, environmental sensing, and biofilm formation.

Visualizing an orphan receptor GPR158 and it complex with neuronal regulator RGS7‐Gβ5 via cryoEM

G protein‐coupled receptors (GPCR) form the largest family of proteins (∼800 GPCRs) encoded in mammalian genomes that detect extracellular signals to program cellular response. They are essential to understanding physiology, disease, and drug development. More than 100 GPCRs are still awaits to identify its endogenous ligand and hence classified as orphan GPCRs. However, these orphan GPCRs have been implicated in several disorders from cancers to neurological diseases. Yet, in many cases, their mechanisms, ligands, and signaling reactions are poorly understood. One such orphan receptor in nervous system is GPR158 that is highly expressed in the brain where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanism of GPR158 are largely unknown. We employed single‐particle cryogenic electron microscopy (cryoEM) to obtain structures of GPR158 in the apo state, and in complex with RGS7‐Gβ5, a regulator of GPCR signaling. The structure reveals highly unique domain organization of GPR158, not documented previously in any GPCRs structures. The homodimeric organization of protomers, phospholipids interaction and the presence of an extracellular Cache domain, an unusual ligand‐binding domain in GPCRs provide insights into the unusual biology of this orphan receptor and the formation of GPCR‐RGS complexes. These structures could help to deorphanize the receptor by serving the template for structure‐based discovery of its ligands that would leads to exploration of mechanistic of GPR158 mediated neuronal signaling.

Cryo-EM structure of human GPR158 receptor coupled to the RGS7-Gβ5 signaling complex.

GPR158 is an orphan G protein–coupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryo–electron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gβ5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.

Phylogenetic Analysis with Prediction of Cofactor or Ligand Binding for Pseudomonas aeruginosa PAS and Cache Domains.

ABSTRACTPAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture.IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

An optimized content caching strategy for video stream in edge-cloud environment

With the mobile data traffic grows rapidly and the video data has a high proportion, the backhaul link faces great pressure. The conventional centralized architecture has been far from enough to satisfy the user demands. For reducing user response latency and easing backhaul stress, the streaming media contents should be proactively stored in the edge of network and new contents caching model needs to be presented. In this work, a video content collaborative caching strategy in the cloud-edge cooperative environment is proposed. In this strategy, first, the k-means algorithm is used to cluster the edge servers. Then the latency and caching cost gain bring by caching the content on the edge servers in the cluster and the collaborative cache domain are analyzed to establish the content caching problem. Further, the marginal gain is calculated by analyzing the latency and caching cost gain. Finally, in order to solve the content caching problem, this paper proposed a marginal gain based content caching algorithm. Experimental results prove the effectiveness of the proposed algorithm.

Basing on microRNA-mRNA analysis identifies microRNA in exosomes associated with wound repair of diabetic ulcers

The diabetic ulcer is one of the serious complications of diabetes. In this study, we aimed to establish an exosomal microRNA (miRNA)-targeted messenger RNA (mRNA) regulatory network for screening new biomarkers for diabetic ulcer treatment. For this purpose, exosomes were extracted from bone marrow stem cells (BMSCs) collected from diabetic ulcer patients and healthy adults. The miRNAs in exosomes was detected by high-throughput sequencing analysis. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential miRNAs were performed. The miRNA-mRNA regulatory network between candidate miRNAs and their target genes were constructed by Cytoscape software basing on mRNA expression profiles data of diabetic ulcer patients from Gene Expression Omnibus (GEO). GO and KEGG analyses of the core genes were performed. A total of 63 differential expressed miRNAs in BMSCs exosomes were identified between diabetic ulcer patients and healthy adults. The GO analysis of miRNAs showed that it was mainly related to signal transduction and intercellular transport, and KEGG analysis showed that it was related to the vascular endothelial growth factor (VEGF) signaling pathway. The core genes of the miRNA-mRNA network were thioredoxin interacting protein (TXNIP), cell division cycle 14A (CDC14A), cache domain containing 1 (CACHD1), interferon-induced protein 44 like (IFI44L), late cornified envelope 1AL (CE1A), leucine-rich repeats and immunoglobulin-like domains 2 (LRIG2), palmdelphin (PALMD) and serine and arginine-rich splicing factor 11 (SRSF11). GO analysis of the core genes was related to platelet-derived growth factor receptor signaling pathway. The KEGG analysis of the core genes was related to the cell cycle and nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. A potential miRNA-mRNA regulatory network provides a comprehensive understanding of the molecular mechanisms and promising new targets such as miR-130a-5p, SESN2, LRIG2, and CDC14A for the wound repair of diabetic ulcers.

Structural analysis of CACHE domain of the McpA chemoreceptor from Leptospira interrogans

Leptospira is a genus of spirochete bacteria highly motile that includes pathogenic species responsible to cause leptospirosis disease. Chemotaxis and motility are required for Leptospira infectivity, pathogenesis, and invasion of bacteria into the host. In prokaryotes, the most common chemoreceptors are methyl-accepting chemotaxis proteins that have a role play to detect the chemical signals and move to a favorable environment for its survival. Here, we report the first crystal structure of CACHE domain of the methyl-accepting chemotaxis protein (McpA) of L. interrogans. The structural analysis showed that McpA adopts similar α/β architecture of several other bacteria chemoreceptors. We also found a typical dimerization interface that appears to be functionally crucial for signal transmission and chemotaxis. In addition to McpA structural analyses, we have identified homologous proteins and conservative functional regions using bioinformatics techniques. These results improve our understanding the relationship between chemoreceptor structures and functions of Leptospira species.

The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Investigating the Role of the Med Protein in Biofilm Formation

Bacillus subtilis is a soil‐dwelling bacterium that serves as a good model organism for the study of different adaptive responses to various environmental conditions. These adaptive responses, including spore formation, cannibalism, and biofilm formation, are controlled by the master regulator Spo0A whose phosphorylation is under the control of five kinases. Initial interest in the protein Med came from its role in the transcription of operons related to cannibalism, and studies show the effect of Med is dependent on one of the five kinases, KinD. These same studies suggest Med may play a role in biofilm formation in a KinD‐dependent manner. The goal of our study of the Med protein is to determine whether it plays a direct role in biofilm formation by directly detecting environmental signals or indirectly through interactions with KinD. To determine whether Med is involved in detecting environmental signals, Δmed and ΔkinD bacteria were compared to wildtype B. subtilis using luciferase reporter assays that quantify the effect of different small molecules on the transcription of biofilm genes. Luciferase assays were compared to biofilm assays under the same conditions to visualize the effect on the biofilm phenotypes. We find that Δmed mutants had a defect in biofilm formation and reduced transcription of biofilm genes compared with wildtype bacteria. However, Δmed mutants were the same as wildtype bacteria in terms of their pattern of stimulation by small molecules. These results indicate that Med is not directly sensing environmental signals and instead is likely to be involved in the biofilm formation pathway indirectly through KinD, which has been shown to bind small molecules through its extracellular CACHE domain. We therefore shifted our focus to investigating interactions between Med and KinD and how Med may affect KinD’s ability to sense small molecules. Med and KinD were purified by tagging them with GST and His tags respectively. Additionally, in the case of KinD we purified constructs of the extracellular and intracellular domains to more precisely determine where the Med protein may be interacting with KinD. Presence of the proteins was determined by SDS‐PAGE and interactions between them was determined by Western blot analysis.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861.

Parallel quorum-sensing system in Vibrio cholerae prevents signal interference inside the host.

Many bacteria use quorum sensing (QS) to regulate virulence factor production in response to changes in population density. QS is mediated through the production, secretion, and detection of signaling molecules called autoinducers (AIs) to modulate population-wide behavioral changes. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified in Vibrio cholerae as QS receptors to activate virulence gene expression at low cell density. Detection of AIs by these receptors leads to virulence gene repression at high cell density. The redundancy among these receptors is puzzling since any one of the four receptors is sufficient to support colonization of V. cholerae in the host small intestine. It is believed that one of the functions of such circuit architecture is to prevent interference on any single QS receptor. However, it is unclear what natural molecules can interfere V. cholerae QS and in what environment interference is detrimental. We show here mutants expressing only CqsR without the other three QS receptors are defective in colonizing the host large intestine. We identified ethanolamine, a common intestinal metabolite that can function as a chemical source of QS interference. Ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR and induces a premature QS response in V. cholerae mutants expressing only CqsR without the other three QS receptors. The effect of ethanolamine on QS gene expression and host colonization is abolished by mutations that disrupt CqsR signal sensing. V. cholerae defective in producing ethanolamine is still proficient in QS, therefore, ethanolamine functions only as an external cue for CqsR. Our findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.

Mobile Edge Cache Strategy Based on Neural Collaborative Filtering

In order to effectively reduce the network transmission delay and improve the network transmission quality, the concept of Content Delivery Network (CDN) is brought forth to provide necessary technical support. In this paper, the edge cooperative caching (ECC) based on machine learning and greedy algorithm is put forward. To start with, the neural collaborative filtering is used to design the content popularity prediction algorithm to realize more accurate prediction of content popularity. Following that, the greedy algorithm after optimization is used to obtain the content delivery strategy of various servers in the cooperative cache domain. Finally, the ECC is adopted to achieve the optimization goal of minimal average content transmission delay. Meanwhile, the simulation experiment is carried out to verify the performance of the ECC. The experimental results suggest that the ECC can effectively improve the cache hit rate and the content cache space utilization, and shorten the average content transmission delay.

The α2δ-like Protein Cachd1 Increases N-type Calcium Currents and Cell Surface Expression and Competes with α2δ-1

SummaryVoltage-gated calcium channel auxiliary α2δ subunits are important for channel trafficking and function. Here, we compare the effects of α2δ-1 and an α2δ-like protein called Cachd1 on neuronal N-type (CaV2.2) channels, which are important in neurotransmission. Previous structural studies show the α2δ-1 VWA domain interacting with the first loop in CaV1.1 domain-I via its metal ion-dependent adhesion site (MIDAS) motif and additional Cache domain interactions. Cachd1 has a disrupted MIDAS motif. However, Cachd1 increases CaV2.2 currents substantially (although less than α2δ-1) and increases CaV2.2 cell surface expression by reducing endocytosis. Although the effects of α2δ-1 are abolished by mutation of Asp122 in CaV2.2 domain-I, which mediates interaction with its VWA domain, the Cachd1 responses are unaffected. Furthermore, Cachd1 co-immunoprecipitates with CaV2.2 and inhibits co-immunoprecipitation of α2δ-1 by CaV2.2. Cachd1 also competes with α2δ-1 for effects on trafficking. Thus, Cachd1 influences both CaV2.2 trafficking and function and can inhibit responses to α2δ-1.

CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

A bipartite periplasmic receptor–diguanylate cyclase pair (XAC2383–XAC2382) in the bacterium Xanthomonas citri

The second messenger cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of bacterial lifestyle, controlling several behaviors, including the switch between sessile and motile states. The c-di-GMP levels are controlled by the interplay between diguanylate cyclases (DGCs) and phosphodiesterases, which synthesize and hydrolyze this second messenger, respectively. These enzymes often contain additional domains that regulate activity via binding of small molecules, covalent modification, or protein-protein interactions. A major challenge remains to understand how DGC activity is regulated by these additional domains or interaction partners in specific signaling pathways. Here, we identified a pair of co-transcribed genes (xac2382 and xac2383) in the phytopathogenic, Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), whose mutations resulted in opposing motility phenotypes. We show that the periplasmic cache domain of XAC2382, a membrane-associated DGC, interacts with XAC2383, a periplasmic binding protein, and we provide evidence that this interaction regulates XAC2382 DGC activity. Moreover, we solved the crystal structure of XAC2383 with different ligands, indicating a preference for negatively charged phosphate-containing compounds. We propose that XAC2383 acts as a periplasmic sensor that, upon binding its ligand, inhibits the DGC activity of XAC2382. Of note, we also found that this previously uncharacterized signal transduction system is present in several other bacterial phyla, including Gram-positive bacteria. Phylogenetic analysis of homologs of the XAC2382-XAC2383 pair supports several independent origins that created new combinations of XAC2382 homologs with a conserved periplasmic cache domain with different cytoplasmic output module architectures.

Structural Basis for Polyamine Binding at the dCACHE Domain of the McpU Chemoreceptor from Pseudomonas putida

Many bacteria can move chemotactically to a variety of compounds and the recognition of chemoeffectors by the chemoreceptor ligand binding domain (LBD) defines the specificity of response. Many chemoreceptors were found to recognize different amino and organic acids, but the McpU chemoreceptor from Pseudomonas putida was identified as the first chemoreceptor that bound specifically polyamines. We report here the three-dimensional structure of McpU-LBD in complex with putrescine at a resolution of 2.4 Å, which fitted well a solution structure generated by small-angle X-ray scattering. Putrescine bound to a negatively charged pocket in the membrane distal module of McpU-LBD. Similarities exist in the binding of putrescine to McpU-LBD and taurine to the LBD of the Mlp37 chemoreceptor of Vibrio cholerae. In both structures, the primary amino group of the respective ligand is recognized by hydrogen bonds established by two aspartate and a tyrosine side chain. This feature may be used to predict the ligands of chemoreceptors with unknown function. Analytical ultracentrifugation revealed that McpU-LBD is monomeric in solution and that ligand binding does not alter this oligomeric state. This sensing mode thus differs from that of the well-characterised four-helix bundle domains where ligands bind to two sites at the LBD dimer interface. Although there appear to be different sensing modes, results are discussed in the context of data, indicating that chemoreceptors employ the same mechanism of transmembrane signaling. This work enhances our understanding of CACHE domains, which are the most abundant sensor domains in bacterial chemoreceptors and sensor kinases.