Crystal structures of GMP kinase in complex with ganciclovir monophosphate and Ap5G.

Guanosine monophosphate kinases (GMPK), by catalyzing the phosphorylation of GMP or dGMP, are of dual potential in assisting the activation of anti-viral prodrugs or as candidates for antibiotic strategies. Human GMPK is an obligate step for the activation of acyclic guanosine analogs, such as ganciclovir, which necessitate efficient phosphorylation, while GMPK from bacterial pathogens, in which this enzyme is essential, are potential targets for therapeutic inhibition. Here we analyze these two aspects of GMPK activity with the crystal structures of Escherichia coli GMPK in complex with ganciclovir-monophosphate (GCV-MP) and with a bi-substrate inhibitor, Ap5G. GCV-MP binds as GMP to the GMP-binding domain, which is identical in E. coli and human GMPKs, but unlike the natural substrate fails to stabilize the closed, catalytically-competent conformation of this domain. Comparison with GMP- and GDP-bound GMPK structures identifies the 2'hydroxyl of the ribose moiety as responsible for hooking the GMP-binding domain onto the CORE domain. Absence of this hydroxyl in GCV-MP impairs the stabilization of the active conformation, and explains why GCV-MP is phosphorylated less efficiently than GMP, but as efficiently as dGMP. In contrast, Ap5G is an efficient inhibitor of GMPK. The crystal structure shows that Ap5G locks an incompletely closed conformation of the enzyme, in which the adenine moiety is located outside its expected binding site. Instead, it binds at a subunit interface that is unique to the bacterial enzyme, which is in equilibrium between a dimeric and an hexameric form in solution. This suggests that inhibitors could be designed to bind at this interface such as to prevent nucleotide-induced domain closure. Altogether, these complexes point to domain motions as critical components to be evaluated in therapeutic strategies targeting NMP kinases, with opposite effects depending on whether efficient phosphorylation or inhibition is being sought after.

Structures of escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity.

BACKGROUND: . Nucleoside monophosphate kinases (NMP kinases) catalyze the reversible transfer of a phosphoryl group from a nucleoside triphosphate to a nucleoside monophosphate. Among them, cytidine monophosphate kinase from Escherichia coli has a striking particularity: it is specific for CMP, whereas in eukaryotes a unique UMP/CMP kinase phosphorylates both CMP and UMP with similar efficiency. RESULTS: . The crystal structure of the CMP kinase apoenzyme from E. coli was solved by single isomorphous replacement and refined at 1.75 A resolution. The structure of the enzyme in complex with CDP was determined at 2.0 A resolution. Like other NMP kinases, the protein contains a central parallel beta sheet, the strands of which are connected by alpha helices. The enzyme differs from other NMP kinases in the presence of a 40-residue insert situated in the NMP-binding (NMPbind) domain. This insert contains two domains: one comprising a three-stranded antiparallel beta sheet, the other comprising two alpha helices. CONCLUSIONS: . Two features of the CMP kinase from E. coli have no equivalent in other NMP kinases of known structure. Firstly, the large NMPbind insert undergoes a CDP-induced rearrangement: its beta-sheet domain moves away from the substrate, whereas its helical domain comes closer to it in a motion likely to improve the protection of the active site. Secondly, residues involved in CDP recognition are conserved in CMP kinases and have no counterpart in other NMP kinases. The structures presented here are the first of a new family of NMP kinases specific for CMP.

X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.

The X-ray structure of Mycobacterium tuberculosis TMP kinase at 1.95 A resolution is described as a binary complex with its natural substrate TMP. Its main features involve: (i) a clear magnesium-binding site; (ii) an alpha-helical conformation for the so-called LID region; and (iii) a high density of positive charges in the active site. There is a network of interactions involving highly conserved side-chains of the protein, the magnesium ion, a sulphate ion mimicking the beta phosphate group of ATP and the TMP molecule itself. All these interactions conspire in stabilizing what appears to be the closed form of the enzyme. A complete multialignment of all (32) known sequences of TMP kinases is presented. Subtle differences in the TMP binding site were noted, as compared to the Escherichia coli, yeast and human enzyme structures, which have been reported recently. These differences could be used to design specific inhibitors of this essential enzyme of nucleotide metabolism. Two cases of compensatory mutations were detected in the TMP binding site of eukaryotic and prokaryotic enzymes. In addition, an intriguing high value of the electric field is reported in the vicinity of the phosphate group of TMP and the putative binding site of the gamma phosphate group of ATP.

Evidence for an active-center cysteine in the SH-proteinase alpha-clostripain through use of N-tosyl-L-lysine chloromethyl ketone.

The rapid reaction of alpha-clostripain with tosyl-L-lysine chloromethyl ketone results in a complete loss of activity and in the disappearance of one titratable SH group whereas the number of histidine residues is not affected. Tosyl-L-phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride have no effect on the catalytic activity. From the molar ratio and under the assumption of 1:1 molar interaction, the fully active enzyme has a specific activity of 650-700 units/mg [twice the value proposed by Porter et al. (J. Biol. Chem. 246 (1971) 7675-7682)]. Partial oxidation makes it experimentally impossible to attain this maximal value.

Borrelia burgdorferi uridine kinase: an enzyme of the pyrimidine salvage pathway for endogenous use of nucleotides.

The 621 bp udk gene encoding Borrelia burgdorferi potential uridine kinase, involved in the pyrimidine salvage pathway, was cloned and sequenced. The B burgdorferi protein has a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel. The N-terminal sequence of the protein, Ala-Lys-Ile-Ile, is identical to that predicted but lacks N-terminal methionine. udk is located at around 15 kb from the left telomere and forms an operon with an upstream ORF. A likely hypothesis for the role of the pyrimidine salvage pathway is the sole use of endogenous nucleotides for Borrelia.

Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme.

Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.

alpha-Clostripain. Chemical characterization, activity, and thiol content of the highly active form of clostripain.

A highly active form of clostripain, composed of two polypeptide chains (Mr = 43,000 and 12,500), was isolated by hydrophobic chromatography from the culture medium of Clostridium histolyticum. It differs in amino acid composition, namely in the value for cyst(e)ine, from that previously reported. The analyses of the separated chains are given. Activity is related to the number of free cysteine residues and full activity is obtained only after complete reduction of the disulfide bonds. Specific modifications by sulfhydryl reagents and tosyl lysine chloromethyl ketone of one thiol group, the one implicated in the activity, are reported. High specificity of alpha-clostripain is restricted to arginyl peptide bonds as tested on parvalbumin.

Amino-acid sequences of the active-site sulfhydryl peptide and other thiol peptides from the cysteine proteinase alpha-clostripain.

One free -SH group in the heavy chain of alpha-clostripain reacts rapidly with N-tosyllysine chloromethyl ketone which inactivates the enzyme. Iodoacetic acid also reacts with the thiol group required for enzyme activity but more slowly. A tryptic peptide containing the reactive sulfhydryl group labelled with iodo[1-14C]acetic acid was isolated and determined to be Gln-Ser-Val-Asp-Leu-Leu-Ala-Phe-Asp-Ala-Cys-Met. All other cysteine peptides were isolated from the trypsin hydrolysate of the [14C]carboxymethylated enzyme. Moreover N-terminal and C-terminal sequences of both chains of alpha-clostripain were determined. The sequences representing 20% of the primary structure of alpha-clostripain are not homologous with either other cysteine proteinases or with any other protein structure known to date.

Primary structure of alpha-clostripain light chain.

The primary structure of light chain of alpha-clostripain was determined by sequence analysis of peptides derived from tryptic digests purified by reverse-phase high-performance liquid chromatography. The 22 isolated tryptic peptides were aligned by peptides derived from chymotryptic and staphylococcal V8 proteinase digests. The light chain contains 133 amino acids residues and has a relative molecular mass of 15400. The prediction of its secondary structure is given.

Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms. Resonance assignment, secondary structure and ligand binding.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.

Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase.

Amino acid analysis, HPLC separation of trypsin digests, and sequence analysis showed that the thermosensitivity of the adenylate kinase (EC 2.7.4.3) from Escherichia coli K-12 strain CR341 T28 results from substitution of a serine residue for proline-87 in the wild-type enzyme. This mutation is accompanied by decreased affinity for nucleotide substrates and decreased catalysis. Circular dichroism spectroscopy showed a significant change of the secondary structure. This mainly corresponds to a reduction in alpha-helix content (39%) of mutant protein as compared to wild-type adenylate kinase (50%). Altered conformation of thermosensitive adenylate kinase was also manifested by an increase in susceptibility to proteolysis by trypsin. Ap5A and ATP, known to induce important conformational changes in eukaryotic adenylate kinase(s), protected the mutant enzyme against inactivation by trypsin. This seems to indicate that the "loosening" of the three-dimensional structure of E. coli adenylate kinase by proline----serine substitution is largely compensated for when an enzyme X ATP or enzyme X Ap5A complex is formed.

Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase.

A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized. This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val. The fusion protein was less stable than the native beta-galatosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase. 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase. Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.

Metal chelating properties of adenylate kinase from Paracoccus denitrificans.

Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp. The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms [Spurgin, P., Tomasselli, A.G., and Schiltz, E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the corresponding gene of P. denitrificans, and to characterize the enzyme overproduced in Escherichia coli. The deduced primary structure of adenylate kinase from P. denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding. The iron-chelated form of recombinant P. denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c. In conclusion, the absence of metal in the enzyme isolated from P. denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.

Genetically engineered zinc-chelating adenylate kinase from Escherichia coli with enhanced thermal stability.

In contrast with adenylate kinase from Gram-negative bacteria, the enzyme from Gram-positive organisms harbors a structural Zn2+ bound to 3 or 4 Cys residues in the structural motif Cys-X2-Cys-X16-Cys-X2-Cys/Asp. Site-directed mutagenesis of His126, Ser129, Asp146, and Thr149 (corresponding to Cys130, Cys133, Cys150, and Cys153 in adenylate kinase from Bacillus stearothermophilus) in Escherichia coli adenylate kinase was undertaken for determining whether the presence of Cys residues is the only prerequisite to bind zinc or (possible) other cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stability. All mutants bearing 3 or 4 cysteine residues acquired zinc binding properties. Moreover, the quadruple mutant exhibited a remarkably high thermal stability as compared with the wild-type form with preservation of the kinetic parameters of the parent enzyme.

Structural and energetic factors of the increased thermal stability in a genetically engineered Escherichia coli adenylate kinase.

Several variants of Escherichia coli adenylate kinase, designed to bind a Zn2+ ion, were produced by site-directed mutagenesis. The metal binding and enzymatic properties of the engineered variants have been described (Perrier, V., Burlacu-Miron, S., Bourgeois, S., Surewicz, W. K., and Gilles, A.-M. (1998) J. Biol. Chem. 273, 19097-19101). Here we report the structural properties and stability changes in a 4-Cys variant which binds a Zn2+ ion and has an increased thermal stability. CD studies indicate a very similar secondary structure content in the wild type and the engineered variant. NMR analysis revealed that the topology of the parallel beta-sheet, belonging to the protein core, and of the peripheral antiparallel beta-sheet are also conserved. The small local changes observed in the neighborhood of the substitution sites reflect a more compact state of the metal-binding domain. The Zn2+-bound quadruple mutant shows an increased thermal stability, reflected in a 9 degreesC increase of the mid-temperature of the first cooperative unfolding step. Binding of a bisubstrate analog P1, P5-di(adenosine-5')-pentaphosphate increases, by about 7 degreesC, the midpoint of this transition in both wild type and modified variant. The NMR data suggest that the peripheral domains involved in substrate binding unfold during the first denaturation step. Urea denaturation experiments indicate an increased resistance against chemical unfolding of the Zn2+-binding variant. In contrast, the Gibbs free energy of unfolding (at physiologically relevant conditions) of the quadruple mutant is lower than that of the wild type.

Characterization of metal and nucleotide liganded forms of adenylate kinase by electrospray ionization mass spectrometry.

Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis, and Bacillus stearothermophilus with the bisubstrate nucleotide analog P1,P5-di(adenosine 5')-pentaphosphate and with metal ions (Zn2+ and/or Mg2+) were analyzed by electrospray ionization mass spectrometry. P1,P5-di(adenosine 5')-pentaphosphate. adenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleotide to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2+ chelated form of the enzyme from the gram-positive organisms. In this way, it is possible in a single mass spectrometry experiment to screen metal-chelating adenylate kinases, without use of radioactively labeled compounds. Binding of Mg2+ to enzyme via P1,P5-di(adenosine 5')-pentaphosphate was also demonstrated by mass spectrometry. Although no amino acid side chain in adenylate kinase is supposed to interact with Mg2+, Asp93 in porcine muscle cytosolic enzyme, equivalent to Asp84 in the E. coli adenylate kinase, was proposed to stabilize the nucleotide.Mg2+ complex via water molecules.

Complete amino acid sequence of the collagenase from the insect Hypoderma lineatum.

The primary structure of the Hypoderma lineatum collagenase was determined. Chymotrypsin digestion and thermolysin fragmentation of the chymotryptic core gave 30 and 5 peptides, respectively, accounting for all the residues of the protein. These peptides were aligned with overlapping peptides derived from tryptic and Staphylococcus aureus V8 proteinase digests. Hypoderma collagenase is a serine proteinase composed of 230 amino acids (Mr 25,223). It displays a high degree of sequential homology with the serine proteinases of the trypsin family, especially with another collagenolytic enzyme, the proteinase I of the crab Uca pugilator. The six half-cystinyl residues of Hypoderma collagenase correspond to 6 of the 10 half-cystinyl residues of chymotrypsin, and the residues forming the charge-relay system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions. The prediction of the secondary structure of the collagenase is given.

A new non-heme iron environment in Paracoccus denitrificans adenylate kinase studied by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy.

Adenylate kinase from the Gram-negative bacterium Paracoccus denitrificans (AKden) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of two metals, zinc and iron, each binding most probably to one monomer. Under oxidizing conditions, the electron paramagnetic resonance (EPR) spectrum of AKden at 4.2 K consists of features at g = 9.23, 4.34, 4.21, and 3.68. These features are absent in the ascorbate-reduced protein and are characteristic of a S = 5/2 spin system in a rhombic environment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) center. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm-1) was estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxidized minus reduced) of AKden are similar to those reported for the non-heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AKden was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubredoxin. Binding of cyanide converts the iron from the high-spin (S = 5/2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non-heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2.18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7. 4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) state allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of 14N or 15N nuclei coupled to the low-spin iron after addition of KC14N or KC15N respectively. This demonstrated that iron in AKden has at least one labile coordination position that can be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.