The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit.     

Homodimerization of the Src homology 3 domain of the calcium channel β-subunit drives dynamin-dependent endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.     

Cytoplasmatic domain of Na,K-ATPase α-subunit is responsible for the aggregation of the enzyme in proteoliposomes

We studied the thermal dependence of amide I′ infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes.     

Mutation in the cysteine bridge domain of the γ-subunit affects light regulation of the ATP synthase but not photosynthesis or growth in Arabidopsis

The chloroplast ATP synthase synthesizes ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine disulfide bridge in CF1 gamma-subunit. In order to investigate the function of the redox regulatory domain and the physiological significance of redox modulation for higher plants, we designed four mutations in the redox regulatory domain of the gamma-subunit to create functional mimics of the permanently reduced form of the gamma-subunit. While the inability to reduce the regulatory disulfide results in lower photosynthesis and growth, unexpectedly, the results reported here show that inability to reoxidize the dithiol may not be of any direct detriment to plant photosynthetic performance or growth.     

Effects of the association between the α-subunit thigh and the β-Subunit EGF2 domains on integrin activation and signaling

Integrin bidirectional signaling is mediated by conformational change. It has been shown that the separation of the α- and β-subunit transmembrane/cytoplasmic tails and the lower legs is required for transmitting integrin bidirectional signals across the plasma membrane. In this study, we address whether the separation of the αβ knee is critical for integrin activation and outside-in signaling. By introducing three disulfide bonds to restrict dissociation of the α-subunit thigh domain and β-subunit I-EGF2 domain, we found that two of them could completely abolish integrin inside-out activation, whereas the other could not. This disulfide-bonded mutant, in the context of the activation mutation of the cytoplasmic domain, had intermediate affinity for ligands and was able to mediate cell adhesion. Our data suggest that there exists rearrangement at the interface between the thigh domain and the I-EGF2 domain during integrin inside-out activation. None of the disulfide-bonded mutants could mediate cell spreading upon adhering to immobilized ligands, suggesting that dissociation of the integrin two knees is required for integrin outside-in signaling. Disrupting the interface by introducing a glycan chain into either subunit is sufficient for high affinity ligand binding and cell spreading.     

Is the isolated ligand binding domain a good model of the domain in the native receptor?

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.     

Both the N-terminal fragment and the protein–protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.     

Compound heterozygosity and nonsense mutations in the α1-subunit of the inhibitory glycine receptor in hyperekplexia

The alpha(1)-inhibitory glycine receptor is a ligand-gated chloride channel composed of three ligand-binding alpha1-subunits and two structural beta-subunits that are clustered on the postsynaptic membrane of inhibitory glycinergic neurons. Dominant and recessive mutations in GLRA1 subunits have been associated with a proportion of individuals and families with startle disease or hyperekplexia (MIM: 149400). Following SSCP and bi-directional di-deoxy fingerprinting mutational analysis of 22 unrelated individuals with hyperekplexia and hyperekplexia-related conditions, we report further novel missense mutations and the first nonsense point mutations in GLRA1, the majority of which localise outside the regions previously associated with dominant, disease-segregating mutations. Population studies reveal the unique association of each mutation with disease, and reveals that a proportion of sporadic hyperekplexia is accounted for by the homozygous inheritance of recessive GLRA1 mutations or as part of a compound heterozygote.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

Di-arginine signals and the K-rich domain retain the Ca²⁺ sensor STIM1 in the endoplasmic reticulum

STIM1 is a core component of the store‐operated Ca²⁺‐entry channel involved in Ca²⁺‐signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca²⁺ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di‐lysine ER‐retention signal. This restricts the function of STIM2 as Ca²⁺ sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell‐cycle‐dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine‐rich domain and a di‐arginine ER retention signal. Store‐operated Ca²⁺‐entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca²⁺‐signaling.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

A domain of the α-subunit of rabbit phosphorylase kinase shows homologies with regions of rabbit α-tropomyosin,human EGF receptor,and the α chain of bovine S-100 protein

Sequence comparison of the -subunit of phosphorylase kinase with -tropomyosin revealed 32% identity, and 49% similarity, between the region of -tropomyosin coded by exon 5 and a 39 amino acid segment of the kinase subunit. A subsequence of the -subunit segment and a sequence overlapping the same -subunit region are homologous with: (a) a region of the cytoplasmic domain of EGF receptor (50% identity) and (b) a Ca²⁺-binding domain of the chain of S-100 protei (50% identity) respectively. Statistical analysis shows that these homologies are significant. The biological implication of the above similarities is discussed.     

The β1 domain of protein G can replace the chorismate mutase domain of the T-protein

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Structural and dynamic studies of the γ-M4 trans-membrane domain of the nicotinic acetylcholine receptor

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the γ-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear α-helical conformation with the helix aligned within 15° of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.