EF-hand Research Articles

The S2–S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation

Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2–S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM1234, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM3, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM1234 on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM1234 or CaM3 are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2–S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM3, indicating that these five mutations decrease the inhibitory effect of Ca2+/CaM. The double mutation C156A/R159A decreases Ca2+/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2–S3 loop in CaM regulation.

Systematic Identification of Protein Targets of Sub5 Using Saccharomyces cerevisiae Proteome Microarrays.

Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linear-ized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to trans-locate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facil-itate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related pro-cess of “actin filament-based process”, “actin filament organization”, “actin cortical patch or-ganization”, regulation of “actin filament bundle assembly”. Moreover, the other enriched cat-egories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment anal-ysis shows protein domain enriched in actin binding, i.e., “Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)”. Being consistent with GO analysis, Search Tool for the Re-trieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed ac-tin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein–protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.

The B″-family subunits of protein phosphatase 2A are necessary for in-vitro dephosphorylation of the Arabidopsis mechanosensory transcription factor VIP1

Protein phosphatase 2A (PP2A) B″-family subunits have Ca2+-binding EF-hand motifs and can bind PP2A substrates. Arabidopsis thaliana PP2A B″-family subunits are encoded by six genes, and bind a transcription factor, VIP1. VIP1 is dephosphorylated and nuclear-localized by hypo-osmotic stress. However, whether PP2A B″-family subunits mediate the VIP1 dephosphorylation is unclear. Here, we show by yeast two-hybrid and in vitro pull down assays that Arabidopsis PP2A B″-family subunits bind Arabidopsis PP2A A (scaffold) subunits. We also show that VIP1 dephosphorylation in vitro can be induced by a PP2A B″-family subunit.

Disrupting polycystin-2 EF hand Ca2+ affinity does not alter channel function or contribute to polycystic kidney disease.

ABSTRACTApproximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2. PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage-dependent gating and subsequent desensitization – two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycystin-2 intracellular EF hand is responsible for these forms of channel regulation, and, if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest that additional Ca2+-binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.

OsClo5 functions as a transcriptional co-repressor by interacting with OsDi19-5 to negatively affect salt stress tolerance in rice seedlings.

Caleosins constitute a small protein family with one calcium-binding EF-hand motif. They are involved in the regulation of development and response to abiotic stress in plants. Nevertheless, how they impact salt stress tolerance in rice is largely unknown. Thereby, biochemical and molecular genetic experiments were carried out, and the results revealed that OsClo5 was able to bind calcium and phospholipids in vitro and localized in the nucleus and endoplasmic reticulum in rice protoplasts. At the germination and early seedlings stages, overexpression transgenic lines and T-DNA mutant lines exhibited reduced and increased tolerance to salt stress, respectively, compared with the wild-type. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays demonstrated that the EF-hand motif of OsClo5 was essential for the interactions with itself and OsDi19-5. Yeast one-hybrid, electrophoretic migration shift and dual-luciferase reporter assays identified OsDi19-5 as a transcriptional repressor via the TACART cis-element in the promoters of two salt stress-related target genes, OsUSP and OsMST. In addition, OsClo5 enhanced the inhibitory effect of OsDi19-5 in the tobacco transient system, which was confirmed by qRT-PCR analysis in rice seedlings under salt stress. The collective results deepen the understanding of the molecular mechanism underlying the roles of caleosin in the salt stress response. These findings will also inform efforts to improve salt tolerance of rice.

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A

Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

Search for New Allergens in Lolium perenne Pollen Growing under Different Air Pollution Conditions by Comparative Transcriptome Study.

The relationship between air pollution and the allergenic capacity of pollen is widely accepted, with allergenicity being directly related to air pollution. To our knowledge, this is the first study comparing the differential expression of Lolium perenne pollen genes by RNAseq, in two wild populations with different levels of air pollution. The objective is to search for proteins that are expressed differentially in both situations and to establish a relationship with increased allergenic capacity. Two populations of L. perenne (Madrid and Ciudad Real) have been studied in two consecutive years, under the rationale that overexpressed genes in Madrid, with higher levels of NO2 and SO2, could be a cause for their greater allergenic capacity. Heat shock proteins (HSP), glycoside hydrolases, proteins with leucin-rich repeat motifs, and proteins with EF-HAND motifs were consistently overexpressed in Madrid pollen in the two years studied. Interestingly, some genes were overexpressed only in one of the years studied, such as pectinesterases in the first year, and lipid transfer proteins (LTPs) and thaumatin in the second. Despite the fact that the potential of all these proteins in relation to possible allergies has been reported, this is the first time they are cited as possible allergens of L. perenne. The results found can contribute decisively to the knowledge of the allergens of L. perenne and their relationship with atmospheric pollution, and to the development of much more effective vaccines.

Purification, crystallization and X-ray analysis of Pf-SCP (sarcoplasmic Ca-binding protein), related to storage and transport of calcium in mantle of Pinctada fucata

Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and β = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.

Structural and functional diversity of Entamoeba histolytica calcium-binding proteins.

Entamoeba histolytica (E. histolytica) is an etiological agent of human amoebic colitis, and it causes a high level of morbidity and mortality worldwide, particularly in developing countries. Ca2+ plays a pivotal role in amoebic pathogenesis, and Ca2+-binding proteins (CaBPs) of E. histolytica appear to be a major determinant in this process. E. histolytica has 27-EF-hand containing CaBPs, suggesting that this organism has complex Ca2+ signaling cascade. E. histolytica CaBPs share (29-47%) sequence identity with ubiquitous Ca2+-binding protein calmodulin (CaM); however, they do not show any significant structural similarity, indicating lack of a typical CaM in this organism. Structurally, these CaBPs are very diverse among themselves, and perhaps such diversity allows them to recognize different cellular targets, thereby enabling them to perform a range of cellular functions. The presence of such varied signaling molecules helps parasites to invade host cells and advance in disease progression. In the past two decades, tremendous progress has been made in understanding the structure of E. histolytica CaBPs by using the X-ray or NMR method. To gain greater insight into the structural and functional diversity of these amoebic CaBPs, we analyzed and compiled all the available literature. Most of the CaBPs has about 150 amino acids with 4-EF hand or EF-hand-like sequences, similar to CaM. In a few cases, all the EF-hand motifs are not capable of binding Ca2+, suggesting them to be pseudo EF-hand motifs. The CaBPs perform diverse cellular signaling that includes cytoskeleton remodeling, phagocytosis, cell proliferation, migration of trophozoites, and GTPase activity. Overall, the structural and functional diversity of E. histolytica CaBPs compiled here may offer a basis to develop an efficient drug to counter its pathogenesis.

Calmodulin-Like (CML) Gene Family in Medicago truncatula: Genome-Wide Identification, Characterization and Expression Analysis.

Calcium is an important second messenger in mediating adaptation responses of plants to abiotic and biotic stresses. Calmodulin-like (CML) protein is an important calcium-signaling protein that can sense and decode Ca2+ signal in plants. Medicago truncatula is a model legume plant; however, investigations of MtCML proteins are limited. Using genome analysis and BLAST database searches, fifty MtCML proteins that possess EF-hand motifs were identified. Phylogenetic analysis showed that CML homologs between M. truncatula, Arabidopsis thaliana and Oryza sativa shared close relationships. Gene structure analysis revealed that these MtCML genes contained one to four conserved EF-hand motifs. All MtCMLs are localized to eight chromosomes and underwent gene duplication. In addition, MtCML genes were differentially expressed in different tissues of M. truncatula. Cis-acting elements in promoter region and expression analysis revealed the potential response of MtCML protein to abiotic stress and hormones. The results provide a basis of further functional research on the MtCML gene family and facilitate their potential use for applications in the genetic improvement on M. truncatula in drought, cold and salt stress environments.

Harnessing Environmental Ca2+ for Extracellular Protein Thermostabilization.

Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca2+-mediated stability. A disorder → order transition accompanies the formation of the Ca2+ complex in the folded state and dictates the minimum Ca2+ concentration at which the Ca2+-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca2+-mediated control elements in signal transduction pathways. Ca2+-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

Extent of polymorphism and selection pressure on the Trypanosoma cruzi vaccine candidate antigen Tc24.

IntroductionChagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem in the Americas, and existing drugs have severe limitations. In this context, a vaccine would be an attractive alternative for disease control. One of the difficulties in developing an effective vaccine lies in the high genetic diversity of T. cruzi. In this study, we evaluated the level of sequence diversity of the leading vaccine candidate Tc24 in multiple parasite strains.Methods and ResultsWe quantified its level of polymorphism within and between T. cruzi discrete typing units (DTUs) and how this potential polymorphism is structured by different selective pressures. We observed a low level of polymorphism of Tc24 protein, weakly associated with parasite DTUs, but not with the geographic origin of the strains. In particular, Tc24 was under strong purifying selection pressure and predicted CD8+ T‐cell epitopes were mostly conserved. Tc24 strong conservation may be associated with structural/functional constrains to preserve EF hand domains and their calcium‐binding loops, and Tc24 is likely important for the parasite fitness.DiscussionTogether, these results show that a vaccine based on Tc24 is likely to be effective against a wide diversity of parasite strains across the American continent, and further development of this vaccine candidate should be a high priority.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

The CnB1 p.D102A variant is linked to dilated cardiomyopathy via impaired Calcineurin activity

Background:The role of calcineurin (protein phosphatase 2B (PP2B)) in the pathogenesis of human dilated cardiomyopathy (DCM) has not been fully elucidated. We determined the potential involvement of calcineurin in the pathogenesis of DCM caused by mutations in CnB1, a subunit of calcineurin.Methods: By whole-exome sequencing, we identified a new CnB1 variant in a Han Chinese proband with cardiomyopathy from a 3-generation family with 2 normal individuals and 3 individuals with familial dilated cardiomyopathy. The potential pathogenic variant was validated by Sanger sequencing. We performed functional and mechanistic experiments in a CnB1-knockin (KI) mouse model and at the cellular level.Results: We detected a rare heterozygous CnB1 variant (p.D102A)in a proband with dilated cardiomyopathy. This variant was localized to the EF hand 3 region of CnB1, where no variants have been previously reported. KI mice harboring the p.D102A variant exhibited decreased cardiac function and cardiac dilatation. Immunoblotting, RT-PCR and immunofluorescence results showed decreased cardiomyocyte size and heart failure-related protein expression. A calcineurin activity assay demonstrated decreased calcineurin activity in the KI mice, accompanied by the decreased ability of CnB1 to bind CnA.Conclusions: CnB1 p.D102A is a disease-associated variantthat confers susceptibility to cardiac dilatation. This variant is associated with impaired calcineurin activity and a subsequent decrease in the ability of CnB1 to bind CnA.

Determination and evaluation of secondary structure content derived from calcium-induced conformational changes in wild-type and mutant mnemiopsin 2 by synchrotron-based Fourier-transform infrared spectroscopy

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo-mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin‑calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers.

Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca2+-bound form in the dark to a Mg2+-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3′,5′-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca2+. Substitutions affect residues directly involved in Ca2+ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca2+ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca2+ and blocking the enzyme in a constitutively active state at physiological levels of Ca2+. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca2+-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.

Molecular Basis of S100A1 Activation and Target Regulation Within Physiological Cytosolic Ca2+ Levels.

The S100A1 protein regulates cardiomyocyte function through its binding of calcium (Ca2+) and target proteins, including titin, SERCA, and RyR. S100A1 presents two Ca2+ binding domains, a high-affinity canonical EF-hand (cEF) and a low-affinity pseudo EF-hand (pEF), that control S100A1 activation. For wild-type S100A1, both EF hands must be bound by Ca2+ to form the open state necessary for target peptide binding, which requires unphysiological high sub-millimolar Ca2+ levels. However, there is evidence that post-translational modifications at Cys85 may facilitate the formation of the open state at sub-saturating Ca2+ concentrations. Hence, post-translational modifications of S100A1 could potentially increase the Ca2+-sensitivity of binding protein targets, and thereby modulate corresponding signaling pathways. In this study, we examine the mechanism of S100A1 open-closed gating via molecular dynamics simulations to determine the extent to which Cys85 functionalization, namely via redox reactions, controls the relative population of open states at sub-saturating Ca2+ and capacity to bind peptides. We further characterize the protein's ability to bind a representative peptide target, TRKT12 and relate this propensity to published competition assay data. Our simulation results indicate that functionalization of Cys85 may stabilize the S100A1 open state at physiological, micromolar Ca2+ levels. Our conclusions support growing evidence that S100A1 serves as a signaling hub linking Ca2+ and redox signaling pathways.

CDNA Cloning and Expression Analysis of Troponin C from Gryllus bimaculatus (Orthoptera: Gryllidae)

Few reports have described the effect of starvation on insect muscle physiology and development. This study examined the role of troponin C (TnC) in the skeletal muscles and digestive tracts of the two-spotted cricket (Gryllus bimaculatus; Orthoptera: Gryllidae) during starvation and re-feeding. The TnC gene of G. bimaculatus (GrybiTnC) was cloned and found to encode a 153-amino-acid protein with a theoretical isoelectric point of 4.02 and molecular weight of 17,368.23 Da. Homology analyses revealed that GrybiTnC exhibited high amino acid similarities with TnCs from other insects, showing sequence identities ranging from ∼54 to 75%. GrybiTnC has two Ca2+-binding sites and five EF-hand motifs, similar to most TnC proteins. GrybiTnC gene expression was examined under starvation and re-feeding conditions in three different skeletal muscle and digestive tract regions. During starvation, GrybiTnC gene expression was unchanged in muscle but upregulated in the foregut and hindgut. Whereas GrybiTnC gene expression in muscle was similar under re-feeding and starvation conditions, it was upregulated in the foregut and midgut by re-feeding with carbohydrates and lipids and downregulated in the hindgut by re-feeding with carbohydrates, proteins and lipids.

Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism.

Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P)+-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD+-specific, NAD+/NADP+ dual-specific, and NADP+-specific. We determined in this work that the mitochondrial AtGDH1 isozyme from A. thaliana is NAD+-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo- and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 in the apo-form and in complex with NAD+ at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD+, it forms D3-symmetric homohexamers. A subunit of AtGDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD+ complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD+ hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25°) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.

Subcellular localization of Rap1 GTPase activator CalDAG‐GEFI is orchestrated by interaction of its atypical C1 domain with membrane phosphoinositides

BackgroundThe small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG‐GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. ObjectiveHere, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. MethodsCDGI interaction with phosphoinositides was studied by lipid co‐sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. ResultsLipid co‐sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)‐biphosphate (PIP2) and phosphatidylinositol (3,4,5)‐triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co‐sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. ConclusionOur studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)‐biphosphate and phosphatidylinositol (3,4,5)‐triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.