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BBA - Proteins and Proteomics (v.1747, #1)

Editorial Board (pp. ii).

The SH2 domain: versatile signaling module and pharmaceutical target by Kazuya Machida; Bruce J. Mayer (pp. 1-25).

The Src homology 2 (SH2) domain is the most prevalent protein binding module that recognizes phosphotyrosine. This ∼100-amino-acid domain is highly conserved structurally despite being found in a wide variety proteins. Depending on the nature of neighboring protein module(s), such as catalytic domains and other protein binding domains, SH2-containing proteins play many different roles in cellular protein tyrosine kinase (PTK) signaling pathways. Accumulating evidence indicates SH2 domains are highly versatile and exhibit considerable flexibility in how they bind to their ligands. To illustrate this functional versatility, we present three specific examples: the SAP, Cbl and SOCS families of SH2-containing proteins, which play key roles in immune responses, termination of PTK signaling, and cytokine responses. In addition, we highlight current progress in the development of SH2 domain inhibitors designed to antagonize or modulate PTK signaling in human disease. Inhibitors of the Grb2 and Src SH2 domains have been extensively studied, with the aim of targeting the Ras pathway and osteoclastic bone resorption, respectively. Despite formidable difficulties in drug design due to the lability and poor cell permeability of negatively charged phosphorylated SH2 ligands, a variety of structure-based strategies have been used to reduce the size, charge and peptide character of such ligands, leading to the development of high-affinity lead compounds with potent cellular activities. These studies have also led to new insights into molecular recognition by the SH2 domain.

Keywords: Abbreviations; pTyr; phosphotyrosine; Cdcp; 3′-carboxyl 4′-dicarboxyl phenylalanine; Fcp; 3′-aldehyde 4′-carboxyl phenylalanine; Dpp; 3′, 4′-diphosphonophenylalanine; dpmF; diphosphonomethyl-phenylalanineProtein tyrosine kinase; Signal transduction; SH2 inhibitor; Phosphotyrosine mimetic

Molecular simulation studies of a selenium-containing scFv catalytic antibody that mimics glutathione peroxidase by Yuan Zhang; Ze-Sheng Li; Miao Sun; Qing-Chuan Zheng; Chia-Chung Sun (pp. 27-34).

GPX is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides (ROOH), using Glutathione (GSH) as the reducing substrate. The single-chain Fv fragment of the monoclonal antibody 2F3 (scFv2F3) can be converted into the selenium-containing Se-scFv2F3 by chemical modification of the serine. The new selenium-containing catalytic antibody Se-scFv2F3 acts as a glutathione peroxidase (GPX) mimic with high catalytic efficiency.In order to investigate which residue of scFv2F3 is converted into selenocysteine and to describe the proper reaction site of GSH to Se-scFv2F3, a three-dimensional structure of scFv2F3 is built by means of homology modeling. The 3D model is assessed by molecular dynamics (MD) simulation to determine its stability and by comparison with those of known protein structures. After the serine in the scFv2F3 is modified to selenocysteine, a catalytic antibody (abzyme) is obtained. From geometrical considerations, the solvent-accessible surface of the protein is examined. The computer-aided docking and energy minimization (EM) calculations of the abzyme–GSH complex are then carried out to explore the possible active site of the glutathione peroxidase mimic Se-scFv2F3. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of GPX.

Keywords: Catalytic antibody; Glutathione peroxidase; Homology modeling; Selenium

A new amphipathy scale by F.M. Mazz�; C.A. Fuzo; L. Degr�ve (pp. 35-46).

Two new amphipathy scales elaborated from molecular dynamics data are presented. Their applications contribute for the identification of the hydrophobic or hydrophilic regions in proteins solely from the primary structure. The new amphipathy coefficients (AC) reflect the side chain/solvent molecules configurational energies. A polar (water) and an apolar solvent, CCl₄, were used resulting in the two AC_water and AC_CCl4 scales. These solvents were chosen to simulate the aqueous phases and the transmembrane ambients of cellular membranes where the membrane proteins act. The new amphipathy scales were compared with some previous scales determined by different methods, which were also compared between them, indicating more than 90% of the correlation coefficients are less than 0.9: the scales are strictly dependent on the methodologies used in their determination. The AC_CCl4 scale is related with the size of side chain amino acids while AC_water is related with the hydrophobicity of side chain amino acids. The quality of the scales was confirmed by an example of application where AC_water was able to identify correctly the transmembrane, hydrophobic regions of a membrane protein. These results also indicate that water is an important factor responsible for the tertiary structure of membrane proteins.

Keywords: Amphipathy; Scale; Molecular dynamics

Isolation and molecular characterization of LVP1 lipolysis activating peptide from scorpion Buthus occitanus tunetanus by Nejla Soudani; Jouda Gharbi-Chihi; Najet Srairi-Abid; Claire Martin-El Yazidi; Richard Planells; Alain Margotat; Janine Torresani; Mohamed El Ayeb (pp. 47-56).

LVP1, a novel protein inducing lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 1% of crude venom proteins, with pHi∼6 and molecular mass of 16170 Da. In contrast to well-characterized scorpion toxins, reduction and alkylation of LVP1 revealed an heterodimeric structure. Isolated α and β chains of LVP1 have a respective molecular mass of 8877 and 8807 Da as determined by mass spectrometry. The N-terminal and some internal peptide sequences of LVP1α and β were determined by Edman degradation. The full amino acid sequences of both chains were deduced from nucleotide sequences of the corresponding cDNAs prepared based on peptide sequences and the 3′ and 5′ RACE methodologies. LVP1α and β cDNAs encode a signal peptide of 22 residues and a mature peptide of 69 and 73 residues, respectively. Each mature peptide contains seven cysteines, which are compatible with an interchain disulfide bridge. The cDNA deduced protein structures share a high similarity with those of some Na⁺ channel scorpion toxins. LVP1 was not toxic to mice after intracerebro-ventricular injection. LVP1 stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 of ∼1+0.5 μg/ml. LVP1 subunits did not display any lipolytic activity. As previously described for venom, β adrenergic receptor (β AR) antagonists interfere with LVP1 activity. Furthermore, it is shown that LVP1 competes with [³H]-CGP 12177 (β₁/β₂ antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10⁻⁷ M. These results demonstrate the existence of a new type of scorpion venom nontoxic peptides that are structurally related to Na⁺ channel toxins but can exert a distinct biological activity on adipocyte lipolysis through a β-type adrenoreceptor pathway.

Keywords: Scorpion venom; Buthus occitanus tunetanus; (Bot); Lipolysis activating venom protein (LVP); Heterodimer; cDNA; Adipocyte; Lipolysis; β adrenergic receptor (β AR)

Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis by Lingyi Lynn Deng; Donald E. Humphries; Robert D. Arbeit; Laura E. Carlton; Sandra C. Smole; J. David Carroll (pp. 57-66).

Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-l-alanyl-d-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.

Keywords: Mycobacterium tuberculosis; AIDS; Cell wall; Cell wall hydrolase; Autolysin; N; -acetylmuramoyl-; l; -alanine amidase; N; -Acetylmuramic acid

Detection of hypothetical proteins in 10 individual human tumor cell lines by Leila Afjehi-Sadat; Joo-Ho Shin; Maureen Felizardo; Kiseok Lee; Irene Slavc; Gert Lubec (pp. 67-80).

The search for new structures in tumors by genomics and proteomics methods is a major goal in tumor biology and may lead to the detection of markers or antigens for the generation of tumor vaccines. The aim of this study was to identify proteins that have been predicted so far based upon their nucleic acid sequence only or show poor identity to known proteins in tumor cell lines. Cell lines of neuroblastoma, colorectal, cervix carcinoma, adenocarcinoma of the ovary, lung and breast cancer, promyelocytic leukaemia, rhabdomyosarcoma, osteosarcoma and malignant melanoma were used. Cell lysates were run on 2D gel electrophoresis with subsequent in-gel digestion and MALDI-TOF-TOF analysis. A series of 10 hypothetical proteins (HPs) were observed and three of these proteins, hypothetical protein (Q9BTE6), CGI-83 protein (Q9Y392) and similar to CG11334 (Q9BV20), were so far described in tumors exclusively. The other seven proteins were already detected at the transcriptional level in normal and tumor cell lines or tissues. In conclusion, the three HPs observed in lung cancer and malignant melanoma may be candidates for development of tumor markers and generation of tumor vaccines.

Keywords: Hypothetical protein; Two-dimensional gel electrophoresis; Human tumor cell line; Cancer vaccine; Tumor marker; MALDI

Effect of genetic variation on the thermal stability of human serum albumin by Ulrich Kragh-Hansen; Shiori Saito; Koji Nishi; Makoto Anraku; Masaki Otagiri (pp. 81-88).

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have T_m values (midpoint of denaturation) higher than, and nine have T_m values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have Δ H_v values (van't Hoff enthalpy) higher than, and 14 have Δ H_v values lower than, normal albumin. All types of combinations of positive and negative Δ T_m values and Δ(Δ H_v) values were found. Good linear correlations between mutation-induced changes of α-helical content and Δ(Δ H_v) values, but not Δ T_m values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on T_m and Δ H_v was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive Δ T_m values and negative Δ(Δ H_v) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.

Keywords: Abbreviations; HSA; human serum albumin; Alb; albumin; proAlb; proalbumin; Alb A; normal (wild-type) albumin; CD; circular dichroism; T; _m; midpoint of denaturation; Δ; H; _v; van't Hoff enthalpyHuman serum albumin; Genetic variant; Thermal stability; Midpoint of denaturation; van't Hoff enthalpy; α-Helical content

Expression, purification, crystallization and preliminary X-ray analysis of strictosidine glucosidase, an enzyme initiating biosynthetic pathways to a unique diversity of indole alkaloid skeletons by Leif Barleben; Xueyan Ma; Juergen Koepke; Guohong Peng; Hartmut Michel; Joachim St�ckigt (pp. 89-92).

Strictosidine β-d-glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 monoterpenoid indole alkaloids with an extremely large number of various carbon skeletons, has been functionally expressed in Escherichia coli and purified to homogeneity in mg scale. Crystals suitable for X-ray analysis were found by robot-mediated screening. Using the hanging-drop technique, optimum conditions were 0.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 and PEG 4000 (10%) as precipitant buffer. The crystals of strictosidine glucosidase belong to the space group P42₁2 with unit cell dimensions of a=157.63, c=103.59 Å and diffract X-rays to 2.48-Å resolution.

Keywords: Strictosidine β-; d; -glucosidase; Cloning and purification; Crystallization; X-ray analysis; Rauvolfia serpentina

Human ribosomal protein S13: cloning, expression, refolding, and structural stability by Alexey Malygin; Natalia Parakhnevitch; Galina Karpova (pp. 93-97).

The cDNA of human ribosomal protein S13 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in Escherichia coli cells. Protein accumulated in the form of inclusion bodies was isolated, purified, and refolded by dialysis. The recombinant protein was immunologically reactive, interacting with antiserum against native rpS13. The secondary structure content of the refolded protein was analyzed by means of CD spectroscopy. It was found that 43±5% of amino acids sequence of the protein form α-helices and 11±3% are placed in β-strands that coincides with theoretical predictions. The β-strands seem to be located in the extension regions of the rpS13 and do not have homologuous regions in the structure of rpS15 from Thermus thermophilus, which is a prokaryotic homolog of rpS13. The protein structure is stable at a pH range from 4.0 to 8.0 and at low concentrations of urea (up to 3 M).

Keywords: Human ribosomal protein S13; Expression; Refolding; Secondary structure

Formation of a misfolded conformation during refolding of HRPA1 in the presence of calcium by Ana Sofia L. Carvalho; Maria Teresa Neves-Petersen; Steffen B. Petersen; Maria Raquel Aires-Barros; Eduardo Pinho e Melo (pp. 99-107).

Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca²⁺-depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, I_Ca²⁺, is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence. The fraction of I_Ca²⁺ increases exponentially with increasing calcium concentration. The I_Ca²⁺ state is formed during refolding after calcium binding to the unfolded state, as reconstitution of HRPA1 from its apoprotein reveals that the affinity of the apoprotein to protoporphyrin IX is higher in the presence of calcium. If calcium is added after refolding only, the majority of HRPA1 molecules retain their native conformation, thus confirming the binding of calcium to the unfolded state.

Keywords: Horseradish peroxidase A1; Misfolded state; Unfolding pathway; Calcium ion; Steady state fluorescence; Circular dichroism

Ions and blockers in potassium channels: insights from free energy simulations by V.B. Luzhkov; J. �qvist (pp. 109-120).

Potassium ion channels enable efficient and selective permeation of K⁺ ions across nonpolar biological membranes. Here we review the results of recent free energy calculations related to the permeation of monovalent cations through K⁺ channels and to the channel inhibition by blocker compounds. In particular, the progress in computational studies of the bacterial KcsA channel is discussed.

Keywords: Potassium channel; KcsA; Ion transport; Blocking; Free energy calculation; Molecular dynamics

Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin by Jason W. Lee; Lawrence T. Nakamura; Michael P. Chang; Bernadine J. Wisnieski (pp. 121-131).

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.

Keywords: Abbreviations; DTx; diphtheria toxin; ADPrT; ADP-ribosyltransferase; CRM197; an ADPrT-deficient form of DTx; EF-2; elongation factor-2; TCA; trichloroacetic acid; TCM; 10 mM Tris–HCl, 2 mM Ca; ²⁺; , 3 mM Mg; ²⁺; , pH 7.5; TMn; 10 mM Tris–HCl, 10 mM Mn; ²⁺; , pH 7.5; dsDNA; double-stranded DNA; ssDNA; single-stranded DNARibosome inactivating protein; elongation factor 2; nuclease; ADPr-transferase activity; Pseudomonas exotoxin A; Gelonin; Apoptosis

Expression and spectroscopic characterization of a large fragment of the μ-opioid receptor by Aaron Kerman; Vettai S. Ananthanarayanan (pp. 133-140).

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human μ-opioid receptor. The fragment (‘TM2–3’), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2–3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2–3 in lysophosphatidylcholine micelles showed that TM2–3 adopts ∼50% α-helical structure in this environment, with the remainder consisting of disordered and/or β-structure. This is consistent with the assumption of an α-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2–3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the μ-opioid receptor and, possibly, other G-protein-coupled receptors as well.

Keywords: Opioid receptor; Membrane protein purification; Inclusion body; Circular dichroism; Secondary structure

Corrigendum to “Dihydropyrimidine dehydrogenase: a flavoprotein with four iron–sulfur clusters�? [Biochim. Biophys. Acta 1701 (2004) 61–74] by Klaus D. Schnackerz; Doreen Dobritzsch; Ylva Lindqvist; Paul F. Cook (pp. 141-141).

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BBA - Proteins and Proteomics (v.1747, #1)