EF-hand Research Articles

Unique methionine-aromatic interactions govern the calmodulin redox sensor

Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous targets. Molecular dynamics simulations were used to define the molecular principles that govern how calmodulin is structurally poised to detect and respond to methionine oxidation. We found that calmodulin's open and closed states were preferentially stabilized by unique, redox sensitive, methionine-aromatic interactions. Key methionine-aromatic interactions were coupled to reorientation of EF hand helices. Methionine to glutamine substitutions designed to mimic methionine oxidation strongly altered conformational transitions by modulating the strength of methionine-aromatic interactions. Together, these results suggest a broadly applicable redox sensing mechanism though which methionine oxidation by cellular oxidants alters the strength of methionine-aromatic interactions critical for functional protein dynamics.

Overexpression of TaCML20, a calmodulin-like gene, enhances water soluble carbohydrate accumulation and yield in wheat.

Calcium (Ca2+ ) is a universal messenger that mediates intracellular responses to extracellular stimuli in living organisms. Calmodulin (CaM) and calmodulin-like (CML) proteins are the important Ca2+ sensors in plants that decode Ca2+ -signatures to execute downstream intracellular level responses. Several studies indicate the interlinking of Ca2+ and sugar signaling in plants; however, no genes have been functionally characterized to provide molecular evidence. Our study found that expression of TaCML20 was significantly correlated with water soluble carbohydrate (WSC) concentrations in recombinant inbred lines in wheat. TaCML20 has four EF-hand motifs that may facilitate the binding of Ca2+ . To explore the role of CML20, we generated TaCML20 overexpressing transgenic lines in wheat. These lines accumulated higher WSC concentrations in the shoots, and we also found a significantly increased transcript level of sucrose:sucrose-1-fructosyltransferase (1-SST) in the internodes compared with the control plants. In addition, TaCML20 overexpressing plants showed significantly increased tillers per plant and also increased about 19% of grain weight per plant compared with control plants. The results also suggested a role for TaCML20 in drought stress, as its transcripts significantly increased in the shoots of wild-type plants under water deficit. These results uncovered the role of CML20 in determining multiple traits in wheat.

Diacylglycerol kinase β induces filopodium formation via its C1, catalytic and carboxy-terminal domains and interacts with the Rac1-GTPase-activating protein, β2-chimaerin

The β-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGKβ remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGKβ in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGKβ mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGKβ. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGKβ selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, β2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with α2-chimaerin, but not with α1- or β1-chimaerin. Moreover, DGKβ enhanced the plasma membrane localization of β2-chimaerin. These results suggest that DGKβ plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation.

Preferential Binding of Mg2+ Over Ca2+ to CIB2 Triggers an Allosteric Switch Impaired in Usher Syndrome Type 1J.

Calcium and integrin binding protein 2 (CIB2) shares with the other members of the CIB family the ability to bind Ca2+ and Mg2+ via two functional EF-hand motifs, namely EF3 and EF4. As a cation sensor, CIB2 is able to switch to a conformation likely associated with specific biological functions yet to be clarified. Recent findings demonstrate the involvement of CIB2 in hearing physiology and a single, conservative point mutation (p.E64D) has been related to Usher Syndrome type 1J (USH1J) and non-syndromic hearing loss. We present an exhaustive biochemical and biophysical characterization of human wild type (WT) and E64D CIB2. We found that CIB2 does not possibly work as a calcium sensor under physiological conditions, its affinity for Ca2+ (Kdapp = 0.5 mM) being too low for detecting normal intracellular levels. Instead, CIB2 displays a significantly high affinity for Mg2+ (Kdapp = 290 μM), and it is probably Mg2+ -bound under physiological conditions. At odds with the homologous protein CIB1, CIB2 forms a non-covalent dimer under conditions that mimic the physiological ones, and as such it interacts with its physiological target α7B integrin. NMR spectroscopy revealed a long-range allosteric communication between the residue E64, located at the N-terminal domain, and the metal cation binding site EF3, located at the C-terminal domain. The conservative E64D mutation breaks up such inter-domain communication resulting in the impaired ability of CIB2 to switch to its Mg2+-bound form. The ability to bind the target integrin peptide was substantially conserved for E64D CIB2, thus suggesting that the molecular defect associated with USH1J resides in its inability to sense Mg2+ and adopt the required conformation.

Characterization of Two EF-hand Domain-containing Proteins from Toxoplasma gondii.

The universal role of calcium (Ca2+ ) as a second messenger in cells depends on a large number of Ca2+ -binding proteins (CBP), which are able to bind Ca2+ through specific domains. Many CBPs share a type of Ca2+ -binding domain known as the EF-hand. The EF-hand motif has been well studied and consists of a helix-loop-helix structural domain with specific amino acids in the loop region that interact with Ca2+ . In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF-hand motif. The majority of these genes have not been characterized. We report the characterization of two EF-hand motif-containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca2+ measurements showed that Δtg216620 cells did not respond to extracellular Ca2+ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca2+ influx while TgGT1_280480 may be playing a different role in the rhoptries.

Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies.

Diacylglycerol kinases (DGKs) are lipid kinases that modulate the levels of lipid second messengers, diacylglycerol and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. DGKα consists of the N-terminal regulatory domains including EF-hand motifs and C1 domains, and the C-terminal catalytic domain (DGKα-CD). To date, however, no structures of mammalian DGKs including their CDs have yet been reported, impeding our understanding on the catalytic mechanism of DGKs and the rational structure-based drug design. Here we attempted to produce DGKα-CD or a full-length DGKα using bacterial and baculovirus-insect cell expression system for structural studies. While several DGKα-CD constructs produced using both bacterial and insect cells formed insoluble or soluble aggregates, the full-length DGKα expressed in insect cells remained soluble and was purified to near homogeneity as a monomer with yields (1.3 mg/mL per one L cell culture) feasible for protein crystallization. Following enzymatic characterization showed that the purified DGKα is in fully functional state. We further demonstrated that the purified enzyme could be concentrated without any significant aggregation, and characterized its secondary structure by circular dichroism. Taken together, these results suggest that the presence of N-terminal regulatory domains suppress protein aggregation likely via their intramolecular interactions with DGKα-CD, and demonstrate that the baculovirus-insect cell expression of the full-length form of DGKα, not DGKα-CD alone, represents a promising approach to produce protein sample for structural studies of DGKα. Thus, our study will encourage future efforts to determine the crystal structure of DGK, which has not been determined since it was first identified in 1959.

Abstract 445: Essential Role of the S100A1-C-terminus Domain in Maintaining Cardiac-inotrope S100A1 Protein Stability

Introduction: S100A1 is a type of S100 protein with two EF-hand motif. It has a unique characteristic of interacting with multiple proteins of contractile machinery & mitochondria. S100A1 protein able to govern proper systolic & diastolic performance of cardiomyocyte interacting, respectively, with SERCA2A & RYR2. S100A1 level decreases dramatically as a consequence of cellular cues such as oxidative-stress or activation of cellular proteasomes. Selective reconstitution of reduced S100A1 level by AAV9-gene transfer rescued failing heart. Because of the unique ability of S100A1 to interact with myriads of proteins with multiple function, we aimed to dissect the structure-function relationship of the domains of S100A1. Methods & results: The C-terminus domain of S100A1 (S100A1ct) equally recapitulated the inotropic & other benefits of the S100A1-FL in experimental model of heart failure & identified as the bioactive lead domain of S100A1. To elucidate the function of the S100A1 without the C-terminus, here after, referred as S100A1ΔCT, we adenoviraly expressed S100A1ΔCT, & as a control, either expressing GFP or S100A1-FL. We, inadvertently, discovered that S100A1ΔCT could not be detected at protein level. PCR analysis & sequencing proved the presence S100A1ΔCT mRNA transcript. Bioinformatics revealed the presence of SUMO interaction motif (SIM) with in the C-terminus & multiple ubiquitination residues on S100A1ΔCT, suggesting the SIM domain of the S100A1 is crucial for stability. Inhibition of cellular proteasome lead to the detection of the S100A1ΔCT protein in cells over expressing S100A1ΔCT. We overexpressed S100a1ΔCT, deletion mutants having part of the SIM motif (S100a1-SIM - ), the entire motif (S100a1-SIM + ) & the S100a1-FL. Whereas the overexpression of S100A1-SIM + & S100A1-FL were detected, S100A1ΔCT & S100A1-SIM - could not be detected at protein level. Conclusion: The C-terminus of S100A1 is essential for stability of S100A1, besides, its potent inotropic function. Taken together, we deciphered the previously unrecognized posttranslational modification governed S100A1 regulation, thus providing new insight into the development of novel therapeutic strategies for heart failure via posttranslational manipulation of S100A1.

DjRlc is required for the intestinal regeneration in planarian Dugesia japonica

The myosin regulatory light chain (RLC) proteins play an important role in cellular processes, especially in muscle contraction. The planarian intestine is a fascinating system for studying the organogenesis during regeneration. In this paper, A homolog gene of Rlc, DjRlc, was identified and characterized in Dugesia japonica. The DjRlc sequence analysis revealed that it contains an opening reading frame encoding a putative protein of 175 amino acids with functionally domains that are highly conserved, including an EF-Hand motif and Ca2+ binding sites. Whole mount in situ hybridization showed that DjRlc is predominantly expressed in the intestine of intact and regenerating planarians. The cross sections of planarians revealed that the DjRlc distributes in the muscle of intact planarians. Knockdown of RNA interference of DjRlc by dsRNA-DjRlc affected the intestinal morphology, causing distinct defects in branching morphogenesis. These finding suggest that DjRlc is required for intestinal regeneration.

Blocking the interaction between S100A9 protein and RAGE V domain using S100A12 protein.

The proteins S100A9 and S100A12 are associated with the human S100 calcium-binding protein family. These proteins promote interaction with target proteins and alter their conformation when they bind to calcium ions in EF-hand motifs. The V domain of RAGE (Receptor for Advanced Glycation End products) is crucial for S100A9 binding. The binding of RAGE with S100 family proteins aids in cell proliferation. In this report, we demonstrate that S100A12 protein hinders the binding of S100A9 with the RAGE V-domain. We used fluorescence and NMR spectroscopy to analyze the interaction of S100A9 with S100A12. The binary complex models of S100A9-S100A12 were developed using data obtained from 1H-15N HSQC NMR titrations and the HADDOCK program. We overlaid the complex models of S100A9-S100A12 with the same orientation of S100A9 and the RAGE V-domain. This complex showed that S100A12 protein blocks the interaction between S100A9 and the RAGE V-domain. It means S100A12 may be used as an antagonist for S100A9. The results could be favorable for developing anti-cancer drugs based on S100 family proteins.

Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses

Calcium (Ca2+) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca2+ binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca2+ signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes. We assigned gene family nomenclature, analyzed gene structure, chromosomal location and gene duplication, as well as protein motif organization. The phylogenetic clustering revealed a total of eight subgroups, including one unique clade of VviCaMs distinct from VviCMLs. VviCaMs were found to contain four EF-hand motifs whereas VviCML proteins have one to five. Most of grapevine CML genes were intronless, while VviCaMs were intron rich. All the genes were well spread among the 19 grapevine chromosomes and displayed a high level of duplication. The expression profiling of VviCaM/VviCML genes revealed a broad expression pattern across all grape organs and tissues at various developmental stages, and a significant modulation in biotic stress-related responses. Our results highlight the complexity of CaM/CML protein family also in grapevine, supporting the versatile role of its different members in modulating cellular responses to various stimuli, in particular to biotic stresses. This work lays the foundation for further functional and structural studies on specific grapevine CaMs/CMLs in order to better understand the role of Ca2+-binding proteins in grapevine and to explore their potential for further biotechnological applications.

Calcium-induced structural rearrangements release autoinhibition in the Rap-GEF CalDAG-GEFI

Platelets are recruited to sites of vascular injury, where they are activated and aggregate to form a hemostatic plug. This process requires the activation of the small GTPase Rap1B by its cognate guanine nucleotide exchange factor CalDAG-GEFI. Studies on platelet function suggest that CalDAG-GEFI activity is regulated by changes in cytosolic calcium, but the exact molecular mechanism is poorly understood. Here we show that purified CalDAG-GEFI is autoinhibited and directly regulated by calcium. Substitutions of putative calcium-binding residues within the canonical EF hands of CalDAG-GEFI diminish its capacity to activate Rap1B. Structural differences between active (WT) and inactive (EF hand variant) CalDAG-GEFI protein were determined by hydrogen-deuterium exchange MS. The highest differential rates of deuterium uptake in WT over EF hand variant CalDAG-GEFI were observed in regions within the catalytic Cdc25 domain and a putative autoinhibitory linker connecting the Cdc25 and EF hand domains. Exchange activity in the EF hand variant was fully restored by an additional substitution, valine 406 to glutamate, which is thought to disrupt the interface between the autoinhibitory linker and the Cdc25 domain. Overall, our results suggest a model for how CalDAG-GEFI remains in an autoinhibited state when levels of cytosolic calcium in resting platelets are low. In response to cellular stimulation, calcium mobilization and binding to the EF hands causes conformational rearrangements within CalDAG-GEFI, including the autoinhibitory linker that frees the catalytic surface of CalDAG-GEFI to engage and activate Rap1B. The data from this study are the first evidence linking CalDAG-GEFI activity directly to calcium.

Genome-wide identification and analysis of MICU genes in land plants and their potential role in calcium stress

Mitochondrial calcium uptake (MICU) plays a vital role in the regulation of mitochondrial calcium homeostasis, and, consequently, influences calcium signaling transduction. Although genes involved in mitochondrial calcium uptake have been well studied in animals, less is known about their ubiquity and function in plants. In this study, we identified 96 MICU genes in land plants. On the basis of phylogenetic analysis of MICU proteins, they were classified into three clades: MICU from eudicots (Clade I), from monocots (Clade II), and from a basal angiosperm, a bryophyte, and a lycophyte (Clade III). Pairwise identity analysis across all MICU proteins showed that they are highly conserved among land plants at the protein level. Conserved motif analysis showed that most MICU proteins contained three EF-hands, and an additional EF-hand motif first identified in the MICU of Arabidopsis thaliana but not mammals was found in all 96 putative MICU proteins. This suggests that a cellular pathway of calcium uptake and signaling that requires three EF-hand motifs is evolutionarily conserved in plants. In addition, we discovered that MICU-defective mutants of Arabidopsis thaliana exhibited longer roots than wild-type under high calcium stress. Concurrently, the mRNA transcription levels of MICU were decreased under high calcium conditions. These results suggest that loss-of-function mutations of MICU may have potential roles in helping plants resist high calcium stress. This study provides clues to the possible role of plant MICU in mitochondrial calcium uptake, as well as useful information to support further studies on MICU function in plants.

Calcineurin Regulatory Subunit Calcium-Binding Domains Differentially Contribute to Calcineurin Signaling in Saccharomyces cerevisiae.

The protein phosphatase calcineurin is central to Ca2+ signaling pathways from yeast to humans. Full activation of calcineurin requires Ca2+ binding to the regulatory subunit CNB, comprised of four Ca2+-binding EF hand domains, and recruitment of Ca2+-calmodulin. Here we report the consequences of disrupting Ca2+ binding to individual Cnb1 EF hand domains on calcineurin function in Saccharomyces cerevisiae Calcineurin activity was monitored via quantitation of the calcineurin-dependent reporter gene, CDRE-lacZ, and calcineurin-dependent growth under conditions of environmental stress. Mutation of EF2 dramatically reduced CDRE-lacZ expression and failed to support calcineurin-dependent growth. In contrast, Ca2+ binding to EF4 was largely dispensable for calcineurin function. Mutation of EF1 and EF3 exerted intermediate phenotypes. Reduced activity of EF1, EF2, or EF3 mutant calcineurin was also observed in yeast lacking functional calmodulin and could not be rescued by expression of a truncated catalytic subunit lacking the C-terminal autoinhibitory domain either alone or in conjunction with the calmodulin binding and autoinhibitory segment domains. Ca2+ binding to EF1, EF2, and EF3 in response to intracellular Ca2+ signals therefore has functions in phosphatase activation beyond calmodulin recruitment and displacement of known autoinhibitory domains. Disruption of Ca2+ binding to EF1, EF2, or EF3 reduced Ca2+ responsiveness of calcineurin, but increased the sensitivity of calcineurin to immunophilin-immunosuppressant inhibition. Mutation of EF2 also increased the susceptibility of calcineurin to hydrogen peroxide inactivation. Our observations indicate that distinct Cnb1 EF hand domains differentially affect calcineurin function in vivo, and that EF4 is not essential despite conservation across taxa.

S100A16 up‑regulates Oct4 and Nanog expression in cancer stem‑like cells of Yumoto human cervical carcinoma cells

Cancer stem-like cells (CSCs), which possess the ability to self-renewal and are multipotent, are regarded as the cause of tumor formation, recurrence, metastasis and drug resistance. It is necessary to understand the properties of CSCs in order to treat them effectively. It has been previously reported that S100 family proteins, which carry calcium-binding EF-hand motifs and are associated with tumorigenic processes, serve crucial roles in maintaining cancer stem-like properties. S100A16 is upregulated in various types of cancer, including bladder, lung and pancreatic. However, the roles of S100A16 in cancer cells, particularly CSCs, are not clear. The present study investigated the roles of S100A16 in CSCs using the sphere formation assay of Yumoto cells, which are a human cervical carcinoma cell line. The mRNA expression levels were evaluated by reverse transcription-polymerase chain reaction and the protein expression levels were detected by western blot analysis. Following the sphere formation of Yumoto cells, the mRNA and protein expression level of Oct4, Nanog and S100A16 were increased compared with the control cells. Following transfection with S100A16 small interfering RNA (siRNA), the mRNA and protein expression of Oct4 and Nanog were decreased and the spheroid size was significantly decreased in the sphere formation of Yumoto cells compared with control siRNA treated cells. There was no change in the p53 mRNA expression level, whereas the p53 protein expression level, which was decreased by the sphere formation, was recovered by S100A16 knockdown. In addition, the protein expression levels of Oct4 and Nanog, which were increased in the sphere formation, were decreased by the proteasome inhibitor lactacystin. No differences were observed in the S100A16 protein expression between the presence or absence of lactacystin. These results suggest that S100A16 serves an important role in the CSCs of human cervical carcinoma and is a positive regulator of Oct4 and Nanog.

A mongolian pine specific endoplasmic reticulum localized CALMODULIN-LIKE calcium binding protein enhances arabidopsis growth

Stress-adapted wild plants are natural sources of novel genes for molecular breeding. Here, we conducted a transcriptional analysis of Pinus sylvestris var. mongolica Litv, an evergreen pine in northeastern China, to identify a novel CALMODULIN-LIKE protein-encoding gene, PsCML1, no significant homologs found in other plant species. PsCML1 encodes a protein predicted to have a single trans-membrane domain at its N-terminal. Four EF-hand motifs (calcium [Ca]-binding structures) are located at its C-terminal and showed Ca2+-specific affinity in isothermal titration calorimetric analysis. Transient expression of PsCML1 in Nicotiana benthamiana showed that the PsCML1 localizes to the endoplasmic reticulum (ER). Heterologous expression of PsCML1 in Arabidopsis significantly promoted seedling growth, and increased resistance to stress from NaCl and Ca2+ deficiency. The roots of the transgenic seedlings had higher contents of cellulose and pectin, but less hemicellulose than those of the wild type (WT). The biosynthesis of cell wall components is linked with protein glycosylation in the ER and reactive oxygen species (ROS) homeostasis. No significant difference was found in the extent of protein glycosylation between the transgenic and WT plants. However, the transgenic roots had higher steady-state levels of ROS, NADPH oxidase activity, and endo-membrane dynamics than those of the WT. A working model was proposed to delineate the interaction among Ca2+, ROS homeostasis, and cell wall loosening-dependent cell division.

The structural modeling of EF-hand motifs in parvalbumin

Parvalbumin (Parv) is a typical protein with EF-hand motifs that play an important role in many physiological processes. We present a novel free energy to model the skeletal C $$_\alpha $$ chain of the protein from the basic principle of mathematics and physics. Starting from the crystal structure of Parv (PDB code 2PVB), we first analyze the profile of the C $$_\alpha $$ bond and torsion angles over the segment that contains the secondary structures. Then the parameters in the energy function are evaluated for the helix ABCD fragment that contains two EF-hand domains in Parv. Meanwhile an eight-soliton configuration at the energy minimum is constructed to model the conformation of ABCD fragment. The deviation of the conformation constructed from the model away from the crystal structure is as small as 1.28 A. The structural modeling stems from the physical energy, which is a benefit relative to the statistics-based or knowledge-based technologies.

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca2+-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1–101), or its short scaffolding domain (81–101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca2+-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca2+-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca2+-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca2+-signals thereby maintaining efficient phototransduction recovery and light adaptation.

Conformational Regulation of Phospholipase C Enzymes

Phospholipase C (PLC) enzymes, in particular the PLCβ and PLCɛ subfamilies, are essential for normal cellular function in the cardiovascular system where they generate second messengers in response to extracellular signaling that increase calcium levels and activate protein kinase C. Crystal structures of PLCβ have provided some insights into its architecture and regulation, but there is no published structural data for PLCɛ or any catalytically active fragment of the enzyme. We have used small angle X‐ray scattering, electron microscopy, and biochemical assays to begin characterizing the solution structures and regulation of PLCɛ and PLCβ. We find that PLCɛ is conformationally dynamic in solution and that its PH domain is essential for maximum basal activity. We also find PLCβ adopts multiple conformations in solution, wherein its PH domain and EF hands are extended away from the catalytic core. Using site‐directed mutagenesis and functional assays, we find that stabilization of the PH domain in its extended or closed conformation impacts its activity. Taken together, our data support a more complex and global model for PLC regulation, wherein the conformation of the PH domain and EF hands relative to the catalytic core modulates its lipase activity at the membrane.Support or Funding InformationThis work is supported by an American Cancer Society Institutional Research Grant (IRG‐14‐190‐56) to the Purdue University Center for Cancer Research and an American Heart Association Scientist Development Grant 16SDG29930017 to A.M.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conformational Dynamics Contribute to Phospholipase Cβ Activity

Phospholipase Cβ (PLCβ) enzymes hydrolyze the lipid phosphatidylinositol‐4,5‐bisphosphate (PIP2) to produce the second messengers inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). Production of these second messengers leads to many diverse physiological responses, including vascular smooth muscle contraction and inflammation. Previous structural and functional studies of PLCβ have revealed a highly conserved catalytic core that adopts a compact, globular structure and forms the minimal fragment that retains lipase activity. However, a growing body of evidence suggests that the PLCβ PH domain, which interacts with EF hands and TIM barrel of the catalytic core in crystal structures, may be flexible in solution. Using a combination of small angle x‐ray scattering (SAXS), cross‐linking studies, and biochemical assays, we provide the first structural data demonstrating that the PH is in fact conformationally flexible in solution. The PH domain adopts two major states: an open state wherein the PH domain is extended away from the core, and a closed state consistent with the crystal structures. We also provide evidence that these conformational states are functionally significant. These findings provide new evidence of conformational heterogeneity of PLC enzymes in solution and reveal new insights into their roles in cell signaling and cardiovascular disease.Support or Funding InformationAmerican Heart Association Scientist Development Grant 16SDG29920017; Purdue Center for Cancer Research; Showalter Foundation; all to A.M.LThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sarcomeric and nonmuscle α-actinin isoforms exhibit differential dynamics at skeletal muscle Z-lines.

The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.