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BBA - Proteins and Proteomics (v.1753, #2)

Editorial Board (pp. ii).

Human tear viscosity: An interactive role for proteins and lipids by Scott M. Gouveia; John M. Tiffany (pp. 155-163).

Human tear viscosity is poorly understood. Tears need to remain on the ocular surface for lubrication without causing damage to the surface epithelia due to drag when blinking. Whole tears are shear-thinning (non-Newtonian), which cannot be explained by the amount of mucin present, nor by individual proteins. Whole tears minus lipids become Newtonian. Though no free lipids had previously been found in collected tears, tear lipocalin (TL), a major tear protein, is known to bind lipids. In this study, we aimed to confirm whether there are any free lipids in collected tears, and to clarify the combined contribution of tear proteins to viscosity, including experiments on recombinant TL, both without (apo-TL) and with (holo-TL) bound lipid. We also investigated possible oligomer formation by holo- and apo-TL as a mechanism for viscosity using SDS-PAGE and analytical ultracentrifugation (AU). For comparison, we have included results for β-lactoglobulin, a well-characterised lipocalin protein. No free lipids were detected in whole tears. Rheology showed that any protein combination that included lysozyme or lactoferrin was shear-thinning, as was apo-TL, though holo-TL was Newtonian (linear). Results from SDS-PAGE and AU showed apo-TL to be entirely monomeric, but holo-TL showed some dimerization. Both apo- and holo-β-lactoglobulin exhibited a monomer–dimer equilibrium. We conclude that hetero-protein interactions, possibly electrostatic, involving lipid-binding-induced structural changes to TL, significantly contribute to the viscosity of human tears.

Keywords: Analytical ultracentrifugation; β-lactoglobulin; Lipid binding; Protein structure; Tear lipocalin; Viscosity

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii by Kelly Neelon; Harold J. Schreier; Heather Meekins; Patrice M. Robinson; Mary F. Roberts (pp. 164-173).

Methanococcus jannaschii accumulates α- and β-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C₃) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature ( T_m) less than 60 °C, well under the growth temperature of the organism (85 °C). None of the small molecule solutes tested (including the K⁺-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 °C. Instead, protein–protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.

Keywords: Methanococcus jannaschii; Glutamine synthetase; Aspartate transcarbamoylase; α-glutamate; β-glutamate; Protein stabilization

Cytochrome b561 protein family: Expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses by Motonari Tsubaki; Fusako Takeuchi; Nobuyuki Nakanishi (pp. 174-190).

Cytochrome b561 family was characterized by the presence of “b561 core domain�? that forms a transmembrane four helix bundle containing four totally conserved His residues, which might coordinate two heme b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL W on b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D (fungi), E (animals/TSF), F (plants+DoH), and G (SDR2). In group A, both motif 1, {FN(X)HP(X)₂M(X)₂G(X)₅G(X)ALLVYR}, and motif 2, {YSLHSW(X)G}, were identified. These two motifs were also conserved in group B. There was no significant features characteristic to groups C and D. A modified version of motif 1, {LFSWHP(X)₂M(X)₃F(X)₃M(X)EAIL(X)SP(X)₂SS}, was found in group E with a high degree of conservation. Both motif 3, {DP(X)WFY(L)H(X)₃Q}, and motif 4, {K(X)R(X)YWN(X)YHH(X)₂G(R/Y)} ,were found in group F at different regions from those of motifs 1 and 2. The “DoH�? domain common to the NH₂-terminal region of dopamine β-hydroxylase was found to form fusion proteins with the b561 core domains in groups F and G. Based on these results, we proposed a hypothesis regarding structures and functions of the 7 subfamilies of cytochrome b561.

Keywords: Abbreviations; DBH; dopamine β-hydroxylase; PHM; peptidylglycine α-amidating monooxygenase; AsA; ascorbate; MDA; monnodehydroascorbate; TSF; tumor suppression factor; Dcytb; duodenal cytochrome b561; SDR2; stromal cell-derived receptor 2Protein structural motif; Cytochrome b561; Transmembrane electron transfer; Membrane protein; Ascorbate

Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods by Koji Tomoo; Yasunori Matsushita; Hiroyuki Fujisaki; Fumi Abiko; Xu Shen; Taizo Taniguchi; Hiroo Miyagawa; Kunihiro Kitamura; Kin-ichiro Miura; Toshimasa Ishida (pp. 191-208).

Taking advantage of the Trp73 residue located close to the 4E-BP binding site of eIF4E, the interaction between the 4E-BP isoform and eIF4E was investigated by the Trp fluorescence titration method. Although no significant difference was observed among the association constants of three 4E-BP isoforms, the binding preference of 4E-BP2 over 4E-BP1 and -BP3 was shown, probably due to the effect of a 4E-BP2-specific LDRR (60–63) sequence for the binding with eIF4E. By contrast, surface plasmon resonance (SPR) analyses showed the binding preference of 4E-BP1, although the difference among the isoforms was also not significant. This inconsistency with fluorescence analysis likely resulted from the different observation points of the interaction, i.e., local and overall interactions observed by the fluorescence and SPR methods, respectively. To clarify the structural basis for these spectroscopic results, the crystal structure of the ternary complex of m⁷GpppA-eIF4E-4E-BP1 fragment (Thr36–Thr70) was analyzed by the X-ray diffraction method. Crystal structure analysis at 2.1 Å resolution revealed that the 4E-BP1 fragment, assigned to the Pro47–Pro66 peptide moiety, adopted a reverse L-shaped conformation involving the β sheet and α-helical structures and was located at the root of the handle of the temple-bell-shaped eIF4E through hydrophilic and hydrophobic interactions. Based on the observed binding mode, possible interactions with the three 4E-BP isoforms have been discussed. On the other hand, since the crystal structural comparison with the previously determined m⁷GpppA-eIF4E-4E binary complex showed that the docking of the 4E-BP1 fragment does not significantly affect the overall tertiary structure and cap-binding scaffold of eIF4E, the dynamic regulation of the cap-binding of eIF4E by 4E-BP1 was investigated by molecular dynamics (MD) simulations. Consequently, the simulation suggested that (i) the helical region of the 4E-BP1 peptide is important for the binding with eIF4E, (ii) the existence of a cap structure stabilizes the binding of eIF4E with 4E-BP, (iii) the binding of 4E-BP stabilizes the cap-binding pocket of eIF4E, and (iv) the phosphorylation of Ser67 alone does not induce the separation of 4E-BP from eIF4E, but increases the structural rigidity of 4E-BP. These results provide the structural basis for the mRNA cap-binding regulation of eIF4E by 4E-BP.

Keywords: Abbreviations; DTT; dithiothreitol; eIF4E; eukaryotic initiation factor 4E; 4E-BP; eIF4E binding protein; MD; molecular dynamics; m; ⁷; G; 7-methyl guanine; m; ⁷; GDP; 7-methylguanosine 5′-diphosphate; m; ⁷; GTP; 7-methylguanosine 5′-triphosphate; m; ⁷; GpppA; P; ¹; -7-methylguanosine-P; ³; -adenosine-5′,5′-triphosphate; mRNA; messenger RNA; PEG; polyethylene glycol; RMS; root mean square; RU; response unit; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPR; surface plasmon resonanceInitiation factor 4E; 4E-binding protein; mRNA cap structure; Fluorescence; Surface plasmon resonance; X-ray crystal structure; Molecular dynamics simulation

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism by K. Mdluli; M.P.S. Booth; R.L. Brady; G. Rumsby (pp. 209-216).

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The K_m values and specificity constants ( K_cat/ K_M) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K_M values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K_M values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD⁺ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH ( K_M 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.

Keywords: LDHA; LDHB; LDH; Glyoxylate reductase; Kinetics; Glyoxylate

Chlamydia pneumoniae AP endonuclease IV could cleave AP sites of double- and single-stranded DNA by Xipeng Liu; Jianhua Liu (pp. 217-225).

Endonuclease IV gene, the only putative AP endonuclease of C. pneumoniae genome, was cloned into pET28a. Recombinant C. pneumoniae endonuclease I V (CpEndoIV) was expressed in E. coli and purified to homogeneity. CpEndoIV has endonuclease activity against apurinic/apyrimidinic sites (AP sites) of double-stranded (ds) oligonucleotides. AP endonuclease activity of CpEndoIV was promoted by divalent metal ions Mg²⁺ and Zn²⁺, and inhibited by EDTA. The natural (A, T, C and G) and modified (U, I and 8-oxo-G (GO)) bases opposite AP site had little effect on the cleavage efficiency of AP site of ds oligonucleotides by CpEndoIV. However, the CpEndoIV-dependent cleavage of AP site opposite modified base GO was strongly inhibited by Chlamydia DNA glycosylase MutY. Interestingly, the AP site in single-stranded (ss) oligonucleotides was also the effective substrate of CpEndoIV. Similar to E. coli endonuclease IV, AP endonuclease activity of CpEndoIV was also heat-stable to some extent, with a half time of 5 min at 60 �C.

Keywords: C. pneumoniae; Endonuclease IV; AP site

G148–GA3: A streptococcal virulence module with atypical thermodynamics of folding optimally binds human serum albumin at physiological temperatures by David A. Rozak; John Orban; Philip N. Bryan (pp. 226-233).

The third albumin binding domain of streptococcal protein G strain 148 (G148–GA3) belongs to a novel class of prokaryotic albumin binding modules that is thought to support virulence in several bacterial species. Here, we characterize G148–GA3 folding and albumin binding by using differential scanning calorimetry and isothermal titration calorimetry to obtain the most complete set of thermodynamic state functions for any member of this medically significant module. When buffered at pH 7.0 the 46-amino acid alpha-helical domain melts at 72 °C and exhibits marginal stability (15 kJ/mol) at 37 °C. G148–GA3 unfolding is characterized by small contributions to entropy from non-hydrophobic forces and a low Δ C_p (1.1 kJ/(deg mol)). Isothermal titration calorimetry reveals that the domain has evolved to optimally bind human serum albumin near 37 °C with a binding constant of 1.4×10 7 M⁻¹. Analysis of G148–GA3 thermodynamics suggests that the domain experiences atypically small per residue changes in structural dynamics and heat capacity while transiting between folded and unfolded states.

Keywords: Streptococcus; Finegoldia magna; Protein G; Albumin binding; Protein stability; Calorimetry

4-Pyridoxolactonase from a symbiotic nitrogen-fixing bacterium Mesorhizobium loti: Cloning, expression, and characterization by Junichi Funami; Yu Yoshikane; Hitoshi Kobayashi; Nana Yokochi; Baiqiang Yuan; Kozo Iwasaki; Kouhei Ohnishi; Toshiharu Yagi (pp. 234-239).

4-Pyridoxolactonase is involved in the degradation pathway for pyridoxine, a free form of vitamin B₆. The gene (mlr6805) encoding the putative 4-pyridoxolactonase of nitrogen fixing symbiotic microorganism Mesorhizobium loti MAFF303099 has been identified based on the genome database. The gene was cloned and overexpressed in a cotransformant Escherichia coli cell. The recombinant enzyme was dimeric protein and contained one mole of Zn²⁺ per mole of subunit. The enzyme showed about 30% identity with various N-acylhomoserine lactone lactonases and metallo-β-lactamases. The phylogram made with ClustalW shows that 4-pyridoxolactonase makes a cluster with Agrobacterium tumefaciens acyl-homoserine lactone lactonase. The alignment of amino acid sequences suggests that 4-pyridoxolactonase has three histidine residues probably involved in binding of Zn²⁺.

Keywords: 4-Pyridoxolactonase; Mesorhizobium loti; Zn-hydrolase; Molecular cloning; Pyridoxine-degradation pathway

Crystal structure of alkyl hydroperoxide-reductase (AhpC) from Helicobacter pylori by Elena Papinutto; Henry J. Windle; Laura Cendron; Roberto Battistutta; Dermot Kelleher; Giuseppe Zanotti (pp. 240-246).

The AhpC protein from H. pylori, a thioredoxin (Trx)-dependent alkyl hydroperoxide-reductase, is a member of the ubiquitous 2-Cys peroxiredoxins family (2-Cys Prxs), a group of thiol-specific antioxidant enzymes. Prxs exert the protective antioxidant role in cells through their peroxidase activity, whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides (ROOH) are reduced and detoxified (ROOH+2e⁻→ROH+H₂O). In this study AhpC has been cloned and overexpressed in E. coli. After purification to homogeneity, crystals of the recombinant protein were grown. They diffract to 2.95 Å resolution using synchrotron radiation. The crystal structure of AhpC has been determined using the molecular replacement method ( R=23.6%, R_free=25.9%). The model, similar in the overall to other members of the 2-Cys Prx family crystallized as toroide-shaped complexes, consists of a pentameric arrangement of homodimers [(α₂)₅ decamer]. The model of AhpC from H. pylori presents significant differences with respect to other members of the family: apart from some loop regions, α5-helix and the C-terminus is shifted, preventing the C-terminal tail of the second subunit from extending toward this region of the molecule. Oligomerization properties of AhpC have been also characterized by gel filtration chromatography.

Keywords: Alkyl hydroperoxide-reductase; AhpC; Helicobacter pylori; 2-Cys peroxiredoxin; Antioxidant

Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity by Hubert Halimi; Josiane De Caro; Fr�d�ric Carri�re; Alain De Caro (pp. 247-256).

Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T₁₇₃–P₁₈₀, V₁₉₉–A₂₀₇) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P₂₀₈–V₂₂₁ (belonging to the β9 loop) and I₂₄₅–F₂₅₈ (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.

Keywords: Abbreviations; HPL; human pancreatic lipase; HPL(-lid); HPL without lid; DP-HPL; HPL covalently inhibited by diethyl-; p; -nitrophenyl phosphate; pAb; polyclonal antibody; ELISA; enzyme-linked immunosorbent assay; PVC; polyvinyl chlorideSPOTscan; Pancreatic lipase; Polyclonal antibody; Anti-synthetic peptide antibody; Epitope mapping; ELISA

Characterization and crystallization of human DPY-30-like protein, an essential component of dosage compensation complex by Xiuhua Dong; Yong Peng; Ying Peng; Feng Xu; Xiaojing He; Feng Wang; Xiaozhong Peng; Boqin Qiang; Jiangang Yuan; Zihe Rao (pp. 257-262).

Human DPY-30-like is a homolog of C. elegans DPY-30. DPY-30 is an essential component of dosage compensation machinery and loss of dpy-30 activity results in XX-specific lethality. In XO animals, DPY-30 is required for developmental processes other than dosage compensation. In yeast, the homolog of DPY-30, Saf19p, functions as a member of histone 3 lysine 4 methylation complex, which is the key part of epigenetic developmental control. In this report, human DPY-30-like protein was overexpressed and purified with the goal of structure determination. It was crystallized at 291 K in hanging drops by the vapour diffusion technique from a precipitant solution consisting of (NH₄)₂SO₄ (1.5–2.0 M), Tris–HCl (0.1 M, pH 8.0). The crystal diffracted to 2.7 Å resolution at 100 K in-house and belongs to the space group P4₁2₁2 or P4₃2₁2 with unit–cell parameters of a= b=74.5 Å, c=87.0 Å, α= β= γ=90.0°. The asymmetric unit contains two molecules with 49% solvent content. We also analyzed its biochemical and biophysical characterizations. Efforts are now under way to determine the molecular structure of the DPY-30-like. These studies will open a new avenue towards the structure−based functional analysis of human DPY-30-like and dosage compensation machinery.

Keywords: DPY-30; Dosage compensation; X chromosome; C. elegans

Exploring the active site of benzaldehyde lyase by modeling and mutagenesis by Malea M. Kneen; Irina D. Pogozheva; George L. Kenyon; Michael J. McLeish (pp. 263-271).

Benzaldehyde lyase (BAL) is a thiamin diphosphate-dependent enzyme, which catalyzes the breakdown of ( R)-benzoin to benzaldehyde. In essence, this is the reverse of the carboligation reaction catalyzed by benzoylformate decarboxylase (BFD). Here, we describe the first steps towards understanding the factors influencing BFD to form a CC bond under conditions wherein BAL will cleave the same bond. What are the similarities and differences between these two enzymes that result in the different catalytic activities? The X-ray structures of BFD and pyruvate decarboxylase (PDC) were used as templates for modeling benzaldehyde lyase. The model shows that a glutamine residue, Gln113, replaces the active site histidines of BFD and PDC. Replacement of the Gln113 by alanine or histidine reduced the value of k_cat for lyase activity by more than 200-fold. The residues in BFD interacting with the phenyl ring of benzoylformate have similarly positioned counterparts in BAL but Ser26, the residue known to interact with the carboxylate group of benzoylformate, has been replaced by an alanine (Ala28). The BAL A28S variant exhibited 7% of WT activity in the BAL assay but, in the most intriguing result, this variant was able to catalyze the decarboxylation of benzoylformate. Conversely, the BFD S26A variant was unable to cleave benzoin.

Keywords: Thiamin diphosphate; Carboligation; Decarboxylation; Catalysis; Interconversion; (; R; )-benzoin

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BBA - Proteins and Proteomics (v.1753, #2)