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The Journal of Biological Chemistry Nov 2018The epithelial Na channel (ENaC) possesses a large extracellular domain formed by a β-strand core enclosed by three peripheral α-helical subdomains, which have been...
The epithelial Na channel (ENaC) possesses a large extracellular domain formed by a β-strand core enclosed by three peripheral α-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (α, β, or γ). Removing the γ subunit thumb domain had no effect on Na currents when expressed in oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the α or β subunit thumb domain exhibited significantly reduced Na currents along with a large reduction in channel surface expression. Moreover, channels lacking an α or γ thumb domain exhibited a diminished Na self-inhibition response, whereas this response was retained in channels lacking a β thumb domain. In turn, deletion of the α thumb domain had no effect on the degradation rate of the immature α subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature β subunit and mature γ subunit was observed when the β or γ thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the α and γ thumb domains both have important roles in the channel's inhibitory response to external Na Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity.
Topics: Animals; Epithelial Sodium Channels; Gene Expression; Humans; Oocytes; Protein Domains; Protein Subunits; Proteolysis; Xenopus laevis
PubMed: 30228189
DOI: 10.1074/jbc.RA118.003618 -
Journal of Chromatography. A Apr 2014Tag-free proteins or protein complexes represent certainly the most authentic starting points for functional or structural studies. They can be obtained by conventional...
Tag-free proteins or protein complexes represent certainly the most authentic starting points for functional or structural studies. They can be obtained by conventional multi-step chromatography from native or recombinant tag-free sources. Alternatively, they can be expressed and purified using a cleavable N-terminal affinity tag that is subsequently removed by a site-specific protease. Proteolytic tag-removal can also be performed "on-column". We show here that this not only represents a very efficient workflow, but also drastically improves the purity of the resulting protein preparations. Precondition for effective on-column-cleavage is, however, that the tag-cleaving protease does not bind the stationary phase. We introduce scAtg4 and xlUsp2 as very good and bdSENP1, bdNEDP1 as well as ssNEDP1 as ideal proteases for on-column cleavage at 4°C. Four of these proteases (bdSENP1, bdNEDP1, scAtg4, xlUsp2) as well as TEV protease display orthogonal, i.e. mutually exclusive cleavage specificities. We combined these features into a streamlined method for the production of highly pure protein complexes: Orthogonal affinity tags and protease recognitions modules are fused to individual subunits. Following co-expression or in-vitro complex assembly, consecutive cycles of affinity capture and proteolytic release then select sequentially for the presence of each orthogonally tagged subunit, yielding protein complexes of well-defined subunit stoichiometry.
Topics: Chromatography, Affinity; Histidine; Multiprotein Complexes; Peptide Hydrolases; Protein Subunits; Recombinant Proteins; SUMO-1 Protein; Substrate Specificity
PubMed: 24636567
DOI: 10.1016/j.chroma.2014.02.030 -
Scientific Reports Jan 2017Human NAD-dependent isocitrate dehydrogenase existing as the αβγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle,...
Human NAD-dependent isocitrate dehydrogenase existing as the αβγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory β and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αβ and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of αβγ requires the assembly and cooperative function of both heterodimers. αβγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate αβγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αβ. Furthermore, the allosteric activation of αβγ is through the γ subunit not the β subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme.
Topics: Adenosine Diphosphate; Allosteric Regulation; Allosteric Site; Citric Acid; Humans; Isocitrate Dehydrogenase; Protein Binding; Protein Multimerization; Protein Subunits
PubMed: 28139779
DOI: 10.1038/srep41882 -
The Journal of General Physiology Nov 2016The TMEM16 family encompasses Ca-activated Cl channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently...
The TMEM16 family encompasses Ca-activated Cl channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca affinities. The dose-response curve of the heterodimer appears to be a weighted sum of two dose-response curves-one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca activation curve for the heterodimer at a low Ca concentration range ([Ca] < 5 µM) is similar to that of the wild-type channel-the Hill coefficients in both cases are significantly greater than one. This suggests that Ca binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a "double-barrel" pore architecture, similar to that found in CLC channels and transporters.
Topics: Animals; Anoctamin-1; Binding Sites; Calcium; Chloride Channels; HEK293 Cells; Humans; Ion Channel Gating; Mice; Mutation; Protein Binding; Protein Subunits
PubMed: 27799319
DOI: 10.1085/jgp.201611651 -
Nature Structural & Molecular Biology Oct 2011The Saccharomyces cerevisiae proteasome comprises a 19-subunit regulatory particle and a 28-subunit core particle. To be degraded, substrates must cross the core...
The Saccharomyces cerevisiae proteasome comprises a 19-subunit regulatory particle and a 28-subunit core particle. To be degraded, substrates must cross the core particle-regulatory particle interface, a site for complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings, one formed by the six ATPase (Rpt) subunits of the regulatory particle and the other by the seven α subunits of the core particle. The Rpt C termini bind to intersubunit cavities in the α-ring, thus directing core particle gating and proteasome assembly. We mapped the Rpt C termini to the α subunit pockets, using a cross-linking approach that revealed an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2-α4, Rpt6-α3 and Rpt3-α2, whereas on the opposite side, the Rpt1, Rpt4 and Rpt5 tails each cross-link to multiple α pockets. Rpt-core particle cross-links are all sensitive to nucleotides, implying that ATP hydrolysis drives dynamic alterations at the core particle-regulatory particle interface.
Topics: Models, Molecular; Multiprotein Complexes; Proteasome Endopeptidase Complex; Protein Conformation; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 22037170
DOI: 10.1038/nsmb.2147 -
Proceedings of the National Academy of... Mar 2020Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial....
Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and β subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), β15Trp(A12), and β71Phe(E15). We also observe electron density evidence for the distal histidine [α58/β63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in β subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or β subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.
Topics: Carbon Monoxide; Crystallography, X-Ray; Diffusion; Heme; Hemoglobins; Histidine; Humans; Ligands; Models, Molecular; Protein Conformation; Protein Subunits; Recombinant Fusion Proteins
PubMed: 32071219
DOI: 10.1073/pnas.1913663117 -
Traffic (Copenhagen, Denmark) Jun 2011Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three...
Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three additional subunits (Trs65, Trs120 and Trs130) and is required for distinct tethering events at Golgi membranes. It is not clear how TRAPPII assembly is regulated. Here, we show that Tca17 is a fourth TRAPPII-specific component, and that Trs65 and Tca17 interact with distinct domains of Trs130 and make different contributions to complex assembly. Whereas Tca17 promotes the stable association of TRAPPII-specific subunits with the core complex, Trs65 stabilizes TRAPPII in an oligomeric form. We show that Trs85, which was previously reported to be a subunit of both TRAPPI and TRAPPII, is not associated with the TRAPPII complex in yeast. However, we find that proteins related to Trs85, Trs65 and Tca17 are part of the same TRAPP complex in mammalian cells. These findings have implications for models of TRAPP complex formation and suggest that TRAPP complexes may be organized differently in yeast and mammals.
Topics: Animals; Humans; Multiprotein Complexes; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 21453443
DOI: 10.1111/j.1600-0854.2011.01181.x -
Biochemistry Jun 2014Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic...
Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV-visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.
Topics: Actinomycetales; Bacterial Proteins; Catalysis; Hydro-Lyases; Kinetics; Protein Processing, Post-Translational; Protein Subunits; Pyridines; Recombinant Proteins
PubMed: 24914472
DOI: 10.1021/bi500260j -
Nucleic Acids Research Jan 2019BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two...
BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Catalytic Domain; DNA Cleavage; DNA Restriction Enzymes; Escherichia coli; Holoenzymes; Mutation; Peptides; Protein Multimerization; Protein Subunits
PubMed: 30395313
DOI: 10.1093/nar/gky1059 -
American Journal of Physiology.... Aug 2014Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational... (Comparative Study)
Comparative Study
Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression.
Topics: Amino Acid Sequence; Animals; Base Sequence; Biodiversity; Chickens; Computational Biology; Computer Simulation; Databases, Protein; Diptera; Humans; Mice; Models, Biological; Models, Genetic; Molecular Sequence Data; Myosin-Light-Chain Phosphatase; Oligochaeta; Phylogeny; Protein Subunits; Zebrafish
PubMed: 24898838
DOI: 10.1152/ajpregu.00145.2014