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BBA - Proteins and Proteomics (v.1794, #7)

Editorial Board (pp. i).

Modulation of the effects of tropomyosin on actin and myosin conformational changes by troponin and Ca²⁺ by Yurii S. Borovikov ⁎; Olga E. Karpicheva; Stanislava V. Avrova; Charles S. Redwood (pp. 985-994).

The molecular mechanisms by which troponin (TN)–tropomyosin (TM) regulates the myosin ATPase cycle were investigated using fluorescent probes specifically bound to Cys36 of TM, Cys707 of myosin subfragment-1, and Cys374 of actin incorporated into ghost muscle fibers. Intermediate states of actomyosin were simulated by using nucleotides and non-hydrolysable ATP analogs. Multistep changes in mobility and spatial arrangement of SH1 helix of myosin motor domain and actin subdomain-1 during the ATPase cycle were observed. Each intermediate state of actomyosin induced a definite conformational state and specific position of TM strands on the surface of thin filament. TM increased the amplitude of myosin SH1 helix and actin subdomain-1 movements at transition from weak- to strong-binding states shifting to the center of thin filament at strong-binding and to the periphery of thin filament at weak-binding states. TN modulated those movements in a Са²⁺-dependent manner. At high-Ca²⁺, TN enhanced the effect of TM on SH1 helix and subdomain-1 movements by transferring TM further to the center of thin filament at strong-binding states. In contrast, at low-Ca²⁺, TN inhibited the effect of TM movements, “freezing” actin structure in “OFF” state and TM in the position typical for weak-binding states, resulting in disturbing the interplay of actin and myosin.

Keywords: Abbreviations; S1; myosin subfragment-1; TM; tropomyosin; TN; troponin complex; TN-I; inhibitory subunit of troponin; IAEDANS; N-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine; IAF; 5-iodoacetamidofluorescein; NEM; N-ethylmaleimide; pPDM; N,N′-1,4-phenylenedimaleimide; DTT; dithiothreitolTropomyosin; Troponin; F-actin; Myosin; Ghost muscle fiber; Fluorescence polarization

Active site loop dictates the thermodynamics of reduction and ligand protonation in cupredoxins by Gianantonio Battistuzzi; Marco Borsari; Christopher Dennison ⁎; Chan Li; Antonio Ranieri; Marco Sola ⁎; Sachiko Yanagisawa (pp. 995-1000).

The thermodynamics of reduction and His ligand protonation have been determined for a range of loop-contraction variants of the electron transferring type 1 copper protein azurin (AZ). For AZPC, in which the native C-terminal loop containing the Cys, His and Met ligands has been replaced with the shorter sequence from plastocyanin (PC) and AZAMI, in which the even shorter amicyanin (AMI) loop has been inserted, the thermodynamics of reduction match those of the protein whose loop has been introduced which are different to the values for AZ. The enthalpic contribution to His ligand protonation, which is not observed in AZ, is similar in AZAMI and AMI. The thermodynamics of this process in AZPC are more dissimilar to those for PC. In the case of AZAMI-F, a variant possessing the (non natural) minimal loop that can bind a type 1 copper site, the reduction thermodynamics are intermediate between those of AZPC and AZAMI, whilst the thermodynamic data for His ligand protonation are very similar to those for AMI. The results for AZAMI and AZPC are primarily due to protein based enthalpic effects related to the interaction of the metal with permanent protein dipoles from the loop, and to the decreased loop length which favors His ligand protonation in the cuprous proteins. Entropic factors related to loop flexibility have little influence because of constraints imposed by metal coordination and the fact that the introduced loops pack well against the AZ scaffold. Thus, the host scaffold in general plays a minor thermodynamic role in both processes, although for AZAMI-F differences in the first and second coordination spheres influence the thermodynamics of reduction.

Keywords: Abbreviations; Δ; H; °′; _rc; enthalpy change for reduction; Δ; S; °′; _rc; entropy change for reduction; E; °′; standard reduction potential; CV; cyclic voltammetry; PGE; pyrolytic graphite edge electrode; SCE; saturated calomel electrode; SHE; standard hydrogen electrode; Δ; H; °′; _AT; enthalpy change for the acid transition; Δ; S; °′; _AT; entropy change for the acid transition; AZ; Pseudomonas aeruginosa; azurin; AMI; Paracoccus denitrificans; amicyanin; PC; plastocyanin from spinach; AZAMI; AZ mutant in which the C-terminal ligand-containing loop of; Paracoccus denitrificans; amicyanin (AMI) has been introduced; AZPC; AZ mutant in which the ligand-containing loop of spinach plastocyanin (PC) has been introduced; AZAMI-F; AZ variant in which the ligand-containing loop of AMI lacking the Phe residue has been introducedCupredoxin; Reduction potential; Electrochemistry; Thermodynamic parameters; Electron transfer; Conformational transition

Crystallographic and modelling studies on Mycobacterium tuberculosis RuvA by J. Rajan Prabu; S. Thamotharan; Jasbeer Singh Khanduja; Nagasuma R. Chandra; K. Muniyappa; M. Vijayan (pp. 1001-1009).

RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of three new crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures presented here and those reported earlier brings to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in Holliday junction binding. This role along with its role in oligomerization could have important biological implications.

Keywords: Recombination; Branch migration; Holliday junction; DNA binding; Oligomerization; Acidic pin

Identification of polyubiquitin binding proteins involved in NF-κB signaling using protein arrays by Beau J. Fenner; Michael Scannell; Jochen H.M. Prehn ⁎ (pp. 1010-1016).

Attachment of ubiquitin to proteins represents a central mechanism for the regulation of protein metabolism and function. In the NF-kappaB pathway, binding of NEMO to polyubiquitinated substrates initiates the pathway in response to cellular stimuli. Other polyubiquitin binding proteins can antagonize this pathway by competing with NEMO for polyubiquitin. We have used protein arrays to identify polyubiquitin binding proteins that regulate NF-kappaB activity. Using polyubiquitin as bait, protein arrays were screened and polyubiquitin binders identified. Novel polyubiquitin binders AWP1, CALCOCO2, N4BP1, RIO3, TEX27, TTC3, UBFD1 and ZNF313 were identified using this approach, while known NF-kappaB regulators including NEMO, A20, ABIN-1, ABIN-2, optineurin and p62 were also identified. Overexpressed AWP1 and RIO3 repressed NF-kappaB activity in a manner similar to optineurin, while siRNAs directed against AWP1 and RIO3 also reduced NF-κB activity. TNFalpha-dependent degradation of IkappaBalpha was also suppressed by overexpression of AWP1 and RIO3, possibly due to the polyubiquitin binding activity of these proteins. Protein array screening using polyubiquitin enabled rapid identification of many known and novel polyubiquitin binding proteins and the identification of novel NF-kappaB regulators.

Keywords: Polyubiquitin; Ubiquitin; Protein array; NF-kappaB; NEMO; AWP1; RIO3; N4BP1; UBC

Generation of hydrogen peroxide in the melanin biosynthesis pathway by Jose Luis Munoz-Munoz; Francisco García-Molina; Ramón Varón; José Tudela; Francisco García-Cánovas ⁎; Jose Neptuno Rodríguez-López (pp. 1017-1029).

The generation of H₂O₂ in the melanin biosynthesis pathway is of great importance because of its great cytotoxic capacity. However, there is controversy concerning the way in which H₂O₂ is generated in this pathway. In this work we demonstrate that it is generated in a series of chemical reactions coupled to the enzymatic formation of o-quinones by tyrosinase acting on monophenols and o-diphenols and during the auto-oxidation of the o-diphenols and other intermediates in the pathway. The use of the enzymes such as catalase, superoxide dismutase and peroxidase helps reveal the H₂O₂ generated. Based on the results obtained, we propose a scheme of enzymatic and non-enzymatic reactions that lead to the biosynthesis of melanins, which explains the formation of H₂O₂.

Keywords: Hydrogen peroxide; Melanins; o; -diphenols; Monophenols; Tyrosinase

Structural insights into the alanine racemase from Enterococcus faecalis by Amit Priyadarshi; Eun Hye Lee; Min Woo Sung; Ki Hyun Nam; Won Ho Lee; Eunice EunKyeong Kim ⁎; Kwang Yeon Hwang ⁎ (pp. 1030-1040).

Alanine racemase (AlaR) is a bacterial enzyme that belongs to the fold-type III group of pyridoxal 5′-phosphate (PLP)-dependent enzymes. AlaR catalyzes the interconversion betweenl- andd-alanine, which is important for peptidoglycan biosynthesis. This enzyme is common in prokaryotes, but absent in eukaryotes, which makes it an attractive target for the design of new antibacterial drugs. Here, we report the crystal structures of both the apoenzyme and thed-cycloserine (DCS) complex of AlaR from the pathogenic bacterium Enterococcus faecalis v583, at a resolution of 2.5 Å. DCS is a suicide inhibitor of AlaR and, as such, serves as an antimicrobial agent and has been used to treat tuberculosis and urinary tract infection-related diseases, and makes several hydrogen bonds with the conserved active site residues, Tyr44 and Ser207, respectively. The apoenzyme crystal structure of AlaR consists of three monomers in the asymmetric unit, including a polyethylene glycol molecule in the dimer interface that surrounds one of the His 293 residues and also sits close to one side of the His 293 residue in the opposite monomer. Our results provide structural insights into AlaR that may be used for the development of new antibiotics targeting the alanine racemase in pathogenic bacteria.

Keywords: Alanine racemase; d; -cycloserine; PEG; PLP; Enterococcus faecalis

The prominent conformational plasticity of lactoperoxidase: A chemical and pH stability analysis by Barbara Boscolo; Sónia S. Leal; Carlos A. Salgueiro; Elena M. Ghibaudi; Cláudio M. Gomes (pp. 1041-1048).

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective C_m values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

Keywords: Abbreviations; LPO; Lactoperoxidase; ESR; Electron Spin Resonance; CD; Circular Dichroism; DSC; Differential Scanning Calorimetry; GdmCl; Guanidinium hydrochloride; MTSL; 1-oxyl-2,2,5,5,-tetramethyl-Δ; ³; -pyrroline-3-methyl)-methanethio-sulfonate spin label; DMF; N,N-Dimethyl Formamide; DMAB; 3-dimethylamino-benzoic acid; MBTH; 3-methyl-2-benzothiazolinone hydrazone hydrochloride; ANS; 1-anilino-naphtalene-8-sulfonateProtein folding; Structural stability; Mammalian peroxidase; Heme protein; Chemical denaturation; pH effect

Mouse aminoacylase 3: A metalloenzyme activated by cobalt and nickel by Kirill Tsirulnikov; Natalia Abuladze; Debra Newman; Sergey Ryazantsev; Talya Wolak; Nathaniel Magilnick; Myong-Chul Koag; Ira Kurtz; Alexander Pushkin ⁎ (pp. 1049-1057).

Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn²⁺-metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained ∼0.35 zinc atoms per monomer. Incubation with Co²⁺ and Ni²⁺ activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn²⁺, Mn²⁺ or Fe²⁺ restored initial activity. Co²⁺ and to a lesser extent Ni²⁺ increased activity several times in comparison with intact wt-AA3. Co²⁺ drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co²⁺ and Ni²⁺.

Keywords: Abbreviations; Ac-DCVC; N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine; Ac-Tyr; N-acetyl-L-tyrosine; AA3; aminoacylase AA3; AA2; aminoacylase 2, aspartoacylase; AA1; aminoacylase 1; CD; circular dichroism; DEAE-cellulose; diethylaminoethyl cellulose; DLS; dynamic light scattering; DTNB; Ellman's reagent, 5,5′-dithio-bis(2-nitrobenzoate); EDTA; ethylenediaminetetraacetic acid; HPLC; high performance liquid chromatography; ICP-MS; inductively coupled plasma mass spectrometry; LC; liquid chromatography; MTSET; [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; MS; mass spectrometry; OP; o; -phenanthroline; PBS; phosphate buffered saline; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCE; trichloroethyleneMetalloprotein; Cobalt; Zinc; Aminoacylase

Role of electrostatic repulsion on colloidal stability of Bacillus halmapalus alpha-amylase by Søren N. Olsen; Kim B. Andersen; Ted W. Randolph; John F. Carpenter; Peter Westh ⁎ (pp. 1058-1065).

The colloidal stability of charged particles in suspension is often controlled by electrostatic repulsion, which can be rationalized in a semi-quantitative way by the DLVO theory. In the current study, we investigate this approach towards understanding irreversible protein aggregation, using Bacillus halmapalus α-amylase (BHA) as a model protein. Repulsive forces between partly unfolded monomers were shown to strongly affect aggregation. Adding salt, increasing valence of counter ions or decreasing pH in the direction of p I resulted in a shift in the rate-limiting step from association to unfolding as evidenced by a change in aggregation kinetics from second to first-order in protein concentration. Charge screening effects by salts resulted in increased average size of protein aggregates but only moderately affected the secondary structure of protein within the aggregates. Salt and pH effects could be explained within the DLVO framework, indicating that partially unfolded BHA monomers can be modelled realistically as colloids with a random charge distribution.

Keywords: Protein aggregation; Aggregation kinetics; Electrostatic repulsion; DLVO theory

A comparative analysis of binding sites between mouse CYP2C38 and CYP2C39 based on homology modeling, molecular dynamics simulation and docking studies by Xuan-Yu Meng; Qing-Chuan Zheng; Hong-Xing Zhang ⁎ (pp. 1066-1072).

Mouse CYP2C38 and CYP2C39 are two closely related enzymes with 91.8% sequence identity. But they exhibit different substrate binding features. In this study, three-dimensional models of CYP2C38 and CYP2C39 were constructed using X-ray crystal structure of human CYP2C8 as the template based on homology modeling methods and molecular dynamics simulations. Tolbutamide as the common substrate of CYP2C38 and CYP2C39 was docked into them and positioned in their active sites with different orientation. All-trans retinoic acid (atRA) is a specific substrate for CYP2C39 and not catalyzed by CYP2C38. By comparison of active site architectures between CYP2C38 and CYP2C39, the possible reasons affecting their substrate binding were proposed. In addition, Arg241, Glu300, Leu366 and Leu476 are identified as critical residue for substrates binding.

Keywords: Cytochrome P450; CYP2C38; CYP2C39; Homology modeling; Molecular dynamics; Molecular docking

Structure and function of a Campylobacter jejuni thioesterase Cj0915, a hexameric hot dog fold enzyme by Takeshi Yokoyama; Kyoung-Jae Choi; Anne M. Bosch; Hye-Jeong Yeo ⁎ (pp. 1073-1081).

Acyl-coenzyme A (CoA) thioesterases are a large family of enzymes that hydrolyze acyl-CoA esters to the free fatty acid and CoA and thereby regulate essential cellular functions such as lipid metabolism, membrane synthesis, signal transduction, and gene transcription. To better understand the virulence mechanisms of Campylobacter jejuni, and its possible link to membrane lipid biosynthesis, we have investigated C. jejuni thioesterases, annotated as putative proteins. While little is known about fatty acid biosynthesis and regulation in C. jejuni, remarkable differences in the genome and its organization from Escherichia coli, the paradigm system, raise questions as to the functions of these putative proteins. Here we present the crystal structure and biochemical analysis of Cj0915, defining the first functional thioesterase from C. jejuni. The structure of Cj0915 reveals the hot dog fold with an YciA-type hexameric assembly. Enzymatic assays performed with the purified protein show that Cj0915 is an efficient thioesterase with a broad specificity toward acyl-CoA substrates. This study provides a framework for investigation on roles of the Cj0915 thioesterase in virulence, and functional activities associated with the Cj0915 thioesterase in vivo.

Keywords: Abbreviations; CoA; coenzyme A; IPTG; isopropyl β-; d; -thiogalactopyranoside; PBS; Phosphate Buffered Saline; GST; Glutathione S-transferase; DTT; dithiothreitol; DTNB; 5,5′-dithio-bis(2-nitrobenzoic acid); HEPES; N; -(2-hydroxyethyl)piperazine-; N; ′-2-ethanesulfonate; HBT; β-hydroxybenzoic acid thioesterasesCrystal structure; Hot dog fold; Thioesterase; Membrane lipid biosynthesis; Campylobacter jejuni

Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases by Francisco Garrido; Carlos Alfonso; John C. Taylor; George D. Markham; María A. Pajares (pp. 1082-1090).

Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of β-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation.

Keywords: Abbreviations; MAT; methionine adenosyltransferase; Mj-MAT; methionine adenosyltransferase from; Methanococcus jannaschii; BsMAT; methionine adenosyltransferase from; Bacillus subtilis; IPTG; isopropyl β-; d; -thiogalactoside; ANS; 8-anilinonaphthalene-1-sulphonic acid; SS; secondary structureS-adenosylmethionine synthetase; Stability; Structural model; Thermophile; Tryptophan mutant; Methionine adenosyltransferase

Marasmius scorodonius extracellular dimeric peroxidase — Exploring its temperature and pressure stability by Matthias Pühse; Renate T. Szweda; Yingying Ma; Christoph Jeworrek; Roland Winter; Holger Zorn ⁎ (pp. 1091-1098).

The temperature and pressure dependent stability and function of MsP1, an uncommon peroxidase from the basidiomycetous fungus Marasmius scorodonius were investigated. To this end, a series of biophysical techniques (DSC, fluorescence and FTIR spectroscopy, small-angle X-ray scattering) were combined with enzymatic studies of the enzyme. The dimeric MsP1 turned out to be not only rather thermostable, but also highly resistant to pressure, i.e., up to temperatures of about 65 °C and pressures as high as 8–10 kbar at ambient temperatures. Remarkably, the activity of MsP1 increased by a factor of two until ∼500 bar. At about 2 kbar, the enzymatic activity was still as high as under ambient pressure conditions. As revealed by the fluorescence and SAXS data, the increased activity of MsP1 at pressures around 500 bar may result from slight structural changes, which might stabilize the transition state of the enzymatic reaction. Owing to this marked high pressure stability of MsP1, it may represent a valuable tool for industrial high pressure applications.

Keywords: Basidiomycete; Peroxidase; High pressure; FTIR; SAXS

Study of a sterol esterase secreted by Ophiostoma piceae: Sequence, model and biochemical properties by Olga Calero-Rueda; Víctor Barba; Enrique Rodríguez; Francisco Plou; Ángel T. Martínez; María Jesús Martínez ⁎ (pp. 1099-1106).

An extracellular sterol esterase from Ophiostoma piceae efficiently hydrolyzes sterol esters, triglycerides and p-nitrophenol esters. cDNA was screened with a probe obtained by PCR using as primers oligonucleotides corresponding to the N-terminal and internal mature enzyme sequences and complete sequence was obtained by 3′ rapid amplification of cDNA end (RACE) and inverse PCR. The O. piceae esterase gene had a length of 1.8 kbp and lacked introns. A search for proteins with related amino acid sequences revealed around 40% identity with lipases from Candida rugosa and Geotrichum candidum. Modelling the O. piceae enzyme, using the crystal structures of Lip1 and Lip3 from C. rugosa as templates, revealed a similar substrate-binding site, but some changes affecting the flap zone and the aromatic region of the tunnel may be responsible for the wide substrate specificity of this interesting sterol esterase. The ability of the new fungal esterase to hydrolyze triglycerides and esters of p-nitrophenol and cholesterol was compared with those of commercial lipases and cholesterol esterases showing the new enzyme the highest efficiency hydrolyzing triglycerides and sterol esters in the conditions assayed (in presence of Genapol X-100). Finally, the O. piceae gene was successfully expressed in Pichia pastoris, as a model organism to express fungal enzymes, resulting in higher levels of esterase activity than those obtained in the O. piceae cultures. In spite of its higher glycosylation degree, the recombinant enzyme was able to hydrolyze more efficiently than native enzyme the assayed substrates.

Keywords: Ophiostoma piceae; Sterol ester; Triglyceride; Wood extractive; Pichia pastoris

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BBA - Proteins and Proteomics (v.1794, #7)