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BBA - Proteins and Proteomics (v.1824, #9)

Editorial Board (pp. i).

The effects of loop size on Sac7d-hairpin DNA interactions by Jeu-Ming P. Yuann; Wen-Hsuan Tseng; Hsin-Ying Lin; Ming-Hon Hou (pp. 1009-1015).

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1–L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA.► Hairpin structure is a common feature of DNA molecules. ► Sac7d binding affinity to hairpin DNA is determined by loop size and stem integrity. ► Tetra-loop is the most favorable one for Sac7d-hairpin DNA interaction. ► For Sac7d-stabilized hairpin DNA, the loop region may absorb this torsional stress.

Keywords: Abbreviations; CD; Circular dichroism; UV; Ultraviolet; SPR; Surface plasmon resonanceDNA-binding protein; Hairpin DNA; Loop size; Stem integrity; Tetra-loop

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens by Lucia Rubio Fernandez; Guy Vandenbussche; Nancy Roosens; Cédric Govaerts; Erik Goormaghtigh; Nathalie Verbruggen (pp. 1016-1023).

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd²⁺, Cu²⁺, Zn²⁺ and Pb²⁺, with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.► A type III MT from Noccaea caerulescens has been expressed and purified. ► The protein binds Cd²⁺, Cu+, Zn²⁺ and Pb²⁺, with different stoichiometries. ► The protein displays a complete absence of periodic secondary structures.

Keywords: Abbreviations; IR; infrared; FTIR; Fourier Transform infrared; ATR; attenuated total reflection; CD; circular dichroism; MT; metallothioneinMetallothionein; Heavy metal; Plant; IR spectroscopy; Metal binding; Mass spectrometry

The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima by Matthew K. Hedge; Alexandra M. Gehring; Chinessa T. Adkins; Leigh A. Weston; Luke D. Lavis; R. Jeremy Johnson (pp. 1024-1030).

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.Display Omitted► Acetyl esterases display a distinct substrate selectivity profile. ► Novel fluorogenic ester substrates provide highly sensitive kinetic measurements. ► Two hydrophobic plasticity residues maintain this unusual substrate specificity. ► Substitution of these plasticity residues imparts broader specificity to TM0077. ► With broad specificity and high stability, TM0077 could be a valuable biocatalyst.

Keywords: Abbreviations; 7-ACA; 7-aminocephalosporanic acid; BTB; bromothymol blue; CE7; carbohydrate esterase family 7; DSF; differential scanning fluorimetry; LB; Luria–Bertani broth; MWCO; molecular weight cut-off; Ni-NTA; nickel-nitrilotriacetic acid; NPA; 4-nitrophenol acetate; PDB; protein data bank; TM0077; acetyl esterase from; Thermotoga maritimaAcetyl esterase; Cephalosporin-C deacetylase; Fluorogenic enzyme substrates; Hydrolases; Substrate specificity

Impact of subunit and oligomeric structure on the thermal and conformational stability of chlorite dismutases by Stefan Hofbauer; Kira Gysel; Georg Mlynek; Julius Kostan; Hagmuller Andreas Hagmüller; Holger Daims; Furtmuller Paul G. Furtmüller; Kristina Djinović-Carugo; Christian Obinger (pp. 1031-1038).

Chlorite dismutases (Cld) are unique heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. Recent phylogenetic and structural analyses demonstrated that these metalloproteins significantly differ in oligomeric and subunit structure. Here we have analyzed two representatives of two phylogenetically separated lineages, namely pentameric Cld from Candidatus “Nitrospira defluvii” and dimeric Cld from Nitrobacter winogradskyi having a similar enzymatic activity at room temperature. By application of a broad set of techniques including differential scanning calorimetry, electronic circular dichroism, UV–vis and fluorescence spectroscopy the temperature-mediated and chemical unfolding of both recombinant proteins were analyzed. Significant differences in thermal and conformational stability are reported. The pentameric enzyme is very stable between pH 3 and 10 ( T_m=92°C at pH 7.0) and active at high temperatures thus being an interesting candidate for bioremediation of chlorite. By contrast the dimeric protein starts to unfold already at 53°C. The observed unfolding pathways are discussed with respect to the known subunit structure and subunit interaction.► Comparison of two chlorite dismutases of different subunit and oligomeric structure but similar enzymatic activity. ► Significant differences in conformational and thermal stability due to different subunit interactions. ► Pentameric (canonical) chlorite dismutase exhibits a high thermal stability and enzymatic activity at high temperatures. ► Presented thermodynamic data are representative for chlorite dismutases of two distinct phylogenetic lineages.

Keywords: Abbreviations; Cld; chlorite dismutase; NdCld; Candidatus; “Nitrospira defluvii”; apo-NdCld; heme-free chlorite dismutase from; Candidatus; “Nitrospira defluvii”; NwCld; chlorite dismutase from; Nitrobacter winogradskyi; DSC; differential scanning calorimetry; ECD; electronic circular dichroism; GdnHCl; guanidinium hydrochlorideChlorite dismutase; Thermal stability; Conformational stability; Protein unfolding; Oligomeric structure; Bioremediation

Structural changes of human RNase L upon homodimerization investigated by Raman spectroscopy by Kriz Martin Kříž; Snasel Jan Snášel; Kopecky Vladimír Kopecký Jr.; Pav Ondřej Páv; Ivan Rosenberg; Stepanek Josef Štěpánek (pp. 1039-1044).

RNase L, a key enzyme in the host defense system, is activated by the binding of 2′–5′-linked oligoadenylates (2–5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2–5 pA₄ and three tetramers with 3′-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA₃X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α–β structural switch may fix domain positions in the hinge region (residues ca. 336–363) during homodimer formation.► Structural response of binding of different pA₃X, including natural one, is uniform. ► Homodimer formation increases β-sheet and decreases α-helical content. ► α–β structural switch probably fixes domain positions during the homodimer formation. ► At least 3 Tyr, 5 Phe and 2 Trp participate in homodimer formation upon 2-5A binding.

Keywords: Abbreviations; 2–5A; 2′–5′-phosphodiester-linked oligoadenylate; AMP; adenosine-5′-phosphate; ANK; ankyrin repeat domain; DCDR; drop coating deposition Raman; DMS; dimethylsuberimidate; LSA; least-squares analysis; PBS; phosphate-buffered saline; PVDF; polyvinylidene difluoride; RIP; reference intensity profiles; RNase L; ribonuclease LRNase L; Raman spectroscopy; DCDR spectroscopy; Phosphonate oligoadenylate; Ligand binding

The C-terminus of human Ca_v2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines by Marcel A. Kamp; Behzad Shakeri; Etienne E. Tevoufouet; Andreas Krieger; Margit Henry; Kerstin Behnke; Stefan Herzig; Jürgen Hescheler; Kayalvizhi Radhakrishnan; Lucie Parent; Toni Schneider (pp. 1045-1057).

Ca_v2.3 containing voltage-activated Ca2⁺ channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca2⁺ which can both, facilitate and inhibit the influx of Ca2⁺ ions through Ca_v2.3. The facilitated Ca2⁺ influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca2⁺ mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca_v2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca_v2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.► Novel calmodulin splice variant (CaM-2-ext) with an extended amino-terminus detected. ► Transcripts of CaM-2-ext are expressed in two tested human cell lines in low amounts. ► The N-terminal extension is hydrophobic, to keep CaM-2-ext membrane-associated. ► CaM-2-ext coprecipitates with overexpressed C-terminus of Cav2.3 in HEK-293 cells. ► CaM-2-ext but not canonical CaM interacts functionally with Cav2.3 in HEK-293 cells.

Keywords: Abbreviations; Ca; _v; 2.3; ion conducting α1 subunit of E-type voltage-gated Ca; 2; ⁺; channel; CDF; Ca; 2; ⁺; dependent facilitation; CDI; Ca; 2; ⁺; dependent inactivation; FLAG; a fusion tag consisting of eight amino acids (DYKDDDDK) including an enterokinase-cleavage site; HVA; high-voltage activated; myc; a fusion tag consisting of 11 amino acids (EQKLISEEDL); LTD; long term depression; LTP; long term potentiation; PKC; protein kinase C; RT; reverse transcription; VGCC; voltage-gated Ca; 2; ⁺; channelProtein–protein interaction; Toxin-resistant calcium current; R-type; HEK-293T cell; hMTC cell; Cap-independent translation

Proteomic analysis of the soluble and the lysosomal+mitochondrial fractions from rat pancreas: Implications for cerulein-induced acute pancreatitis by Garcia-Hernandez Violeta García-Hernández; Sanchez-Bernal Carmen Sánchez-Bernal; Nancy Sarmiento; Raúl A. Viana; Laura Ferreira; Perez Nieves Pérez; José J. Calvo; Sanchez-Yague Jesús Sánchez-Yagüe (pp. 1058-1067).

Alterations in protein expression within the initiation phase of acute pancreatitis (AP) might play an important role in the development of this disease, lysosomes being involved in its pathophysiology. The use of pancreatic subcellular fractions in proteomic analysis, simplifies protein maps and helps in the identification of new protein changes and biomarkers characterizing tissue damage. The present study aims to determine the differentially expressed acidic proteins in the pancreatic soluble and lysosomal+mitochondrial (L+M) fractions from rats during the early phase of the experimental model of cerulein (Cer)-induced AP. Subcellular pancreatic extracts from diseased and control rats were analyzed by 2-DE (3–5.6 pH range) and MALDI-TOF/TOF MS. Comparative analysis afforded the conclusive identification of 13 (soluble fraction) and 7 (L+M fraction) proteins or protein fragments ocurring in different amounts between diseased and control pancreas, some of them being newly described in AP. In the soluble fraction, we detected changes related to inflammation and apoptosis (α1-inhibitor-3, α-1 antitrypsin, α-1 macroglobulin, haptoglobin, STRAP), oxidative stress and stress response (peroxiredoxin-2, thioredoxin-like 1, GRP94/TRA1, heat shock cognate 71kDa protein), digestive proteases (elastase 3B), serine protease inhibition (serpins B6 and A3L) and translation processes (EF 1-δ). In the L+M fraction, we detected changes mainly related to energy generation or cellular metabolism (ATP synthase β subunit, chymotrypsinogen B, triacylglycerol lipase), cell redox homeostasis (iodothyronine 5´monodeiodinase) and digestive proteases (carboxypeptidase B1). The data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP.► Pancreatic 2DE-analysis during the early phase of experimental acute pancreatitis. ► Subcellular fractionation helps in the identification of new acidic protein changes. ► New described up-regulated proteins: STRAP, serpin B6 and thioredoxin-like 1. ► Overexpressed proteins in the lysosomal+mitochondrial fraction: ATP5B, CTRB1. ► Iodothyronine 5´monodeiodinase decreased in the lysosomal+mitochondrial fraction.

Keywords: Abbreviations; AAT; α1-antitrypsin; ACN; acetonitrile; AP; acute pancreatitis; ASK1; apoptosis signal-regulating kinase 1; ATP5B; ATP synthase beta chain; CBP; carboxypeptidase; Cer; cerulein; CTRB1; chymotrypsinogen B; L; +; M; lysosomal; +; mitochondrial fraction; STRAP; Ser/Thr receptor-associated protein; TGF-β; transforming growth factor β; TXNL-1; thioredoxin-like protein 1Acute pancreatitis; Cerulein; Two-dimensional gel electrophoresis; Proteomics; Lysosome

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BBA - Proteins and Proteomics (v.1824, #9)