Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.     

Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly

Pierisin-1, a cytotoxic protein found naturally in the cabbage butterfly, induces apoptosis of mammalian cells. Our recent studies suggest that pierisin-1 consists of an N-terminal ADP-ribosyltransferase domain, and a C-terminal region that binds to receptors on the surfaces of target cells and incorporates the protein into cells. The present study was undertaken to identify receptors for pierisin-1. The cross-linking and cloning experiments suggested that the proteins on cell membrane had no binding ability to pierisin-1. Inhibitory assays of fractionated lipids from human cervical carcinoma HeLa cells, which are highly sensitive to pierisin-1, indicated neutral glycosphingolipids on the cell surface to show receptor activity. Inhibitory assays and TLC immunostaining using anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids as active components. Analysis of their structures with glycosphingolipid-specific antibodies and negative secondary ion mass spectrometry identified them as globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4 for pierisin-1 were also confirmed with these authentic compounds. Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1 receptors. The C-terminal region of pierisin-1 consists of possible lectin domains of a ricin B-chain, containing QXW sequences, which are essential for its structural organization. Alteration of QXW by site-directed mutagenesis caused marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal region, in a manner similar to ricin, and then exhibits cytotoxicity after incorporation into the cell.     

Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (epsilon) subunit of Escherichia coli DNA polymerase III

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH <6.5 and 400 min(-1) at pH >9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.     

Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels

Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca(2+) binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1-22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca(2+) dissociation rate constants k(3,4), 1.7+/-0.5 s(-1)) than to the N-lobe (k(1,2), 10.8+/-1.3 s(-1)). In contrast, peptides representing the C-terminal tail domain (residues 208-227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k(3,4), 2.6+/-0.1 s(-1) or k(1), 13.8+/-0.5 s(-1) and k(2), 1000 s(-1)). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.     

Exosite interactions impact matrix metalloproteinase collagen specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.     

Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

Proteolysis by plasmin inactivates bovine ADP-ribosyltransferase; therefore, enzymatic activity depends exclusively on the intact enzyme molecule. The transferase was hydrolyzed by plasmin to four major polypeptides, which were characterized by affinity chromatography and N-terminal sequencing. Based on the cDNA sequence for human ADP-ribosyltransferase enzyme [Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., & Sugimura, T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622], a polypeptide map of the bovine enzyme was constructed by superposing the experimentally determined N-terminal sequences of the isolated polypeptides on the human sequence deduced from its cDNA. Two polypeptides, the N-terminal peptide (Mr 29,000) and the polypeptide adjacent to it (Mr 36,000), exhibited binding affinities toward DNA, whereas the C-terminal peptide (Mr 56,000), which accounts for the rest of the transferase protein, bound to the benzamide-Sepharose affinity matrix, indicating that it contains the NAD+-binding site. The fourth polypeptide (Mr 42,000) represents the C-terminal end of the larger C-terminal fragment (Mr 56,000) and was formed by a single enzymatic cut by plasmin of the polypeptide of Mr 56,000. The polypeptide of Mr 42,000 still retained the NAD+-binding site. The plasmin-catalyzed cleavage of the polypeptide of Mr 56,000-42,000 was greatly accelerated by the specific ligand NAD+. Out of a total of 96 amino acid residues sequenced here, there were only 6 conservative replacements between human and bovine ADP-ribosyltransferase.     

The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration. The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1). Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest. It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.     

Bacillus sphaericus mosquitocidal toxin (MTX) and pierisin: the enigmatic offspring from the family of ADP-ribosyltransferases

The mosquitocidal toxin (MTX) from Bacillus sphaericus and the apoptosis-inducing pierisin-1 from the cabbage butterfly Pieris rapae are two of the most intriguing members of the family of ADP-ribosyltransferases. They are both approximately 100 kDa proteins, composed of an N-terminal ADP-ribosyltransferase (approximately 27 kDa) and a C-terminal putative binding and translocation domain (approximately 70 kDa) consisting of four ricin-B-like domains. While they both share structural homologies, with an overall amino acid sequence identity of approximately 30% that becomes approximately 50% at the level of the catalytic core, and functional similarities, notably in terms of enzyme regulation, they seem to largely differ with regard to their targets or cell internalization mechanisms. MTX ADP-ribosylates numerous proteins in lysates of target insect cells at arginine residues, whereas pierisin-1 modifies DNA of insect and mammalian cells by ADP-ribosylation at 2'-deoxyguanosine residues resulting in DNA adducts, mutations and eventually apoptosis. This target specificity differentiates pierisin-1 from all other ADP-ribosyltransferases described so far, and implies that the enzyme must reach the nucleus of target cells. The recently solved crystal structure of MTX catalytic domain is helpful to reveal new insights into structural organization, catalytic mechanisms, proteolytic activation and autoinhibition of both enzymes. The uptake and processing of the ADP-ribosyltransferases is discussed.     

Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae.

The cabbage butterfly Pieris rapae contains a strong apoptosis-inducing substance, pierisin, against human cancer cell lines, which is thought to act via ADP-ribosylation. Here we report the purification and cloning of an apoptosis-inducing substance, designated as pierisin-2, from another cabbage butterfly, Pieris brassicae. Pierisin-2 was purified from pupae by sequential chromatography and its cytotoxic and apoptosis-inducing activities to various cancer cells were similar to those of pierisin, designated as pierisin-1, from P. rapae. cDNA cloning of pierisin-2 was performed on the basis of the partial amino-acid sequence. The nucleotide sequence indicated that the cDNA encodes an 850-amino-acid protein with a calculated molecular mass of 97 986. The deduced amino-acid sequence of pierisin-2 was 91% identical with that of pierisin-1. In vitro expressed protein in the reticulocyte lysate exhibited apoptosis-inducing activities against human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, similar to the purified native pierisin-2 from the pupae. Pierisin-2 shows regional sequence similarities with certain ADP-ribosylating toxins such as the A-subunit of cholera toxin. The results from site-directed mutagenesis at Glu165, a conserved residue among ADP-ribosylating enzymes necessary for NAD binding, and from experiments with ADP-ribosylating enzyme inhibitors suggested that pierisin-2 could be considered as an ADP-ribosylating toxin like pierisin-1.     

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.     

Characterization of Saccharomyces cerevisiae Atm1p: functional studies of an ABC7 type transporter

Saccharomyces cerevisiae Atm1p has been cloned, over-expressed and purified from a yeast expression system. The sequence includes both the soluble ATPase and transmembrane-spanning domains. With the introduction of an N-terminal Kozak sequence and a C-terminal (His)(6)-tag, a yield of 1 mg of Atm1p was obtained from 3 g wet yeast cells, which is comparable to other membrane-associated proteins isolated from eukaryotic expression systems. The ATPase activity of Atm1p is sensitive to sodium vanadate, a P-type ATPase inhibitor, with an IC(50) of 4 microM. MgADP is a product inhibitor for Atm1p with an IC(50) of 0.9 mM. The Michaelis-Menten constants V(max), K(M) and k(cat) of Atm1p were measured as 8.7+/-0.3 microM/min, 107+/-16 microM and 1.24+/-0.06 min(-1), respectively. A plot of ATPase activity versus concentration of Atm1p exhibits a nonlinear relationship, suggesting an allosteric response and an important role for the transmembrane domain in mediating both ATP hydrolysis and MgADP release. The metal dependence of Atm1p ATPase activity demonstrated a reactivity order of Mg(2+)>Mn(2+)>Co(2+), while each divalent ion was found to be inhibitory at higher concentrations. The activation and inhibitory effect of phospholipids suggest that formation of a lipid-micelle complex is important for enzymatic activity and stability. Structural analysis of Atm1p by CD spectroscopy suggested a similarity of secondary structure to that found for other members of this ABC protein family.     

Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding

The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.     

Identification of N-terminal regions of wheat leaf ferredoxin NADP+ oxidoreductase important for interactions with ferredoxin

Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADP(+) reductase (pFNRI and pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displays N-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation on interaction with ferredoxin (Fd), recombinant pFNRII proteins, differing by deletions of up to 25 amino acids, were generated. During purification of the isoproteins found in vivo, the longer forms of pFNRII bound more strongly to a Fd affinity column than did the shorter forms, pFNRII(ISKK) and pFNRII[N-2](KKQD). Further truncation of the N-termini resulted in a pFNRII protein which failed to bind to a Fd column. Similar k(cat) values (104-140 s(-1)) for cytochrome c reduction were measured for all but the most truncated pFNRII[N-5](DEGV), which had a k(cat) of 38 s(-1). Stopped-flow kinetic studies, examining the impact of truncation on electron flow between mutant pFNRII proteins and Fd, showed there was a variation in k(obs) from 76 to 265 s(-1) dependent on the pFNRII partner. To analyze the sites which contribute to Fd binding at the pFNRII N-terminal, three mutants were generated, in which a single or double lysine residue was changed to glutamine within the in vivo N-terminal truncation region. The mutations affected binding of pFNRII to the Fd column. Based on activity measurements, the double lysine residue change resulted in a pFNRII enzyme with decreased Fd affinity. The results highlight the importance of this flexible N-terminal region of the pFNRII protein in binding the Fd partner.     

The ADP-ribosylating mosquitocidal toxin from Bacillus sphaericus: proteolytic activation, enzyme activity, and cytotoxic effects.

The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a approximately 97-kDa protein sharing sequence homology within the N terminus with the catalytic domains of various bacterial ADP-ribosyltransferases. Here we studied the proteolytic activation of the ADP-ribosyltransferase activity of MTX. Chymotrypsin treatment of the 97-kDa MTX holotoxin (MTX(30-870)) results in a 70-kDa putative binding component (MTX(265-870)) and a 27-kDa enzyme component (MTX(30-264)), possessing ADP-ribosyltransferase activity. Chymotryptic cleavage of an N-terminal 32-kDa fragment of MTX (MTX(30-308)) also yields MTX(30-264), but the resulting ADP-ribosyltransferase activity is much greater than that of the processed MTX(30-870). Kinetic studies revealed a K(m) NAD value of 45 microm for the processed 32-kDa MTX fragment, and a K(m) NAD value of 1300 microm for the processed holotoxin. Moreover, the k(cat) value for the activated MTX(30-308) fragment was about 10-fold higher than that for the activated holotoxin (MTX(30-870)). Precipitation analysis showed that the 70-kDa proteolytic fragment of MTX remains noncovalently bound to the N-terminal 27-kDa fragment, thereby inhibiting ADP-ribosyltransferase and NAD glycohydrolase activities. Glu(197) of MTX(30-264) was identified as the "catalytic" glutamate that is conserved in all ADP-ribosyltransferases. Whereas mutated MTX(30-264)E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activity, mutated MTX(30-264)E195Q possesses glycohydrolase activity but not transferase activity. Transfection of HeLa cells with a vector encoding a fusion protein of MTX(30-264) with a green fluorescent protein led to cytotoxic effects characterized by cell rounding and formation of filopodia-like protrusions. These cytotoxic effects were not observed with the catalytically inactive MTX(30-264)E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.     

CPG70 is a novel basic metallocarboxypeptidase with C-terminal polycystic kidney disease domains from Porphyromonas gingivalis

In a search for a basic carboxypeptidase that might work in concert with the major virulence factors, the Arg- and Lys-specific cysteine endoproteinases of Porphyromonas gingivalis, a novel 69.8-kDa metallocarboxypeptidase CPG70 was purified to apparent homogeneity from the culture fluid of P. gingivalis HG66. Carboxypeptidase activity was measured by matrix-assisted laser desorption ionization-mass spectrometry using peptide substrates derived from a tryptic digest of hemoglobin. CPG70 exhibited activity with peptides containing C-terminal Lys and Arg residues. The k(cat)/K(m) values for the hydrolysis of the synthetic dipeptides FA-Ala-Lys and FA-Ala-Arg by CPG70 were 99 and 56 mm(-1)s(-1), respectively. The enzyme activity was strongly inhibited by the Arg analog (2-guanidinoethylmercapto)succinic acid and 1,10-phenanthroline. High resolution inductively coupled plasma-mass spectrometry demonstrated that 1 mol of CPG70 was associated with 0.6 mol of zinc, 0.2 mol of nickel, and 0.2 mol of copper. A search of the P. gingivalis W83 genomic data base (TIGR) with the N-terminal amino acid sequence determined for CPG70 revealed that the enzyme is an N- and C-terminally truncated form of a predicted 91.5-kDa protein (PG0232). Analysis of the deduced amino acid sequence of the full-length protein revealed an N-terminal signal sequence followed by a pro-segment, a metallocarboxypeptidase catalytic domain, three tandem polycystic kidney disease domains, and an 88-residue C-terminal segment. The catalytic domain exhibited the highest sequence identity with the duck metallocarboxypeptidase D domain II. Insertional inactivation of the gene encoding CPG70 resulted in a P. gingivalis isogenic mutant that was avirulent in the murine lesion model under the conditions tested.     

Calcium binding to calmodulin and its globular domains

The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.     

Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS-PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k(cat)/K(M) of 602,000 M(-1)s(-1), PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.