Structural basis for inactivation of Giardia lamblia carbamate kinase by disulfiram.

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.

The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK) catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK) with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP), was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170), which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.

Correlation of structure and function in the human hotdog-fold enzyme hTHEM4.

Human THEM4 (hTHEM4) is comprised of a catalytically active hotdog-fold acyl-CoA thioesterase domain and an N-terminal domain of unknown fold and function. hTHEM4 has been linked to Akt1 regulation and cell apoptosis. Herein, we report the X-ray structure of hHTEM4 bound with undecan-2-one-CoA. Structure guided mutagenesis was carried out to confirm the catalytic residues. The N-terminal domain is shown to be partially comprised of irregular and flexible secondary structure, reminiscent of a protein-binding domain. We demonstrate direct hTHEM4-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that hTHEM4 is an Akt1 negative regulator.

A homogenous luminescence assay reveals novel inhibitors for giardia lamblia carbamate kinase.

The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z' factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member.

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg(2+)-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, alpha and beta, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GKbeta encodes a peptide that is different in sequence and is 16 amino acids longer than that encoded by the N-terminal exon of GKalpha. The crystal structures of recombinant GKalphabeta and GKbetabeta from Namalycastis sp. were determined at 2.6 and 2.4 A resolution, respectively. In addition, the structure of the GKbetabeta was determined at 2.3 A resolution in complex with a transition state analogue, Mg(2+)-ADP-NO(3)(-)-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GKalphabeta are structurally defined, and the longer N-terminus of the beta subunit is anchored at the dimer interface. In GKbetabeta the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GKalphabeta amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely alpha or solely beta chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.

Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

Swiveling domain mechanism in pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg(2+)-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg(2+) and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg(2+) and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189 approximately P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an alpha-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189 approximately P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

Novel structure and nucleotide binding properties of HI1480 from Haemophilus influenzae: a protein with no known sequence homologues.

The crystal structure of the Haemophilus influenzae protein HI1480 was determined at 2.1-A resolution. The amino acid sequence of HI1480 is unique, having no homology with other known protein sequences. The protein adopts a novel alpha+beta fold, and associates into a dimer of tightly associated dimers. The tight dimers are formed by intermolecular interactions that are mediated by an antiparallel beta-barrel involving both monomers. Helical regions of two dimers mediate the tetramer formation. The helical region contains a four-helix bundle that has been seen only in the anticodon binding domains of class I tRNA synthetases. A cluster of four residues, Tyr18, Arg134, Glu26, and Lys12 is located in a depression formed at the four-helix bundle/ beta-barrel interface. The arrangement is suggestive of an active center, possibly a catalytic site. The HI1480 gene is located within the Mu-like prophage region of H. influenzae, has no homology to bacteriophage genes, and is flanked by transposases. Hence, this is an example of horizontal transfer from an unknown organism. Gel mobility shift assays revealed that HI1480 binds DNA and RNA molecules. Double-stranded DNA is favored over single-stranded DNA, and longer DNA molecules are bound better than shorter ones.

Structural insight into arginine degradation by arginine deiminase, an antibacterial and parasite drug target.

l-Arginine deiminase (ADI) catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia. ADI is involved in the first step of the most widespread anaerobic route of arginine degradation. ADI, missing in high eukaryotes, is a potential antimicrobial and antiparasitic drug target. We have determined the crystal structure of ADI from Pseudomonas aeruginosa by the multi-wavelength anomalous diffraction method at 2.45 A resolution. The structure exhibits similarity to other arginine-modifying or substituted arginine-modifying enzymes such as dimethylarginine dimethylaminohydrolase (DDAH), arginine:glycine amidinotransferase, and arginine:inosamine-phosphate amidinotransferase, despite the lack of significant amino acid sequence homology to these enzymes. The similarity spans a core domain comprising five betabetaalphabeta motifs arranged in a circle around a 5-fold pseudosymmetry axis. ADI contains an additional alpha-helical domain of novel topology inserted between the first and the second betabetaalphabeta modules. A catalytic triad, Cys-His-Glu/Asp (arranged in a different manner from that of the thiol proteases), seen in the other arginine-modifying enzymes is also conserved in ADI, as well as many other residues involved in substrate binding. Based on this conservation pattern and the assumption that the substrate binding mode is similar to that of DDAH, an ADI catalytic mechanism is proposed. The main players are Cys-406, which mounts the nucleophilic attack on the carbon atom of the guanidinium group of arginine, and His-278, which serves as a general base.

Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot.

The crystal structures of YibK from Haemophilus influenzae (HI0766) have been determined with and without bound cofactor product S-adenosylhomocysteine (AdoHcy) at 1.7 and 2.0 A resolution, respectively. The molecule adopts an alpha/beta fold, with a topology that differs from that of the classical methyltransferases. Most notably, HI0766 contains a striking knot that forms the binding crevice for the cofactor. The knot formation is correlated with an alternative arrangement of the secondary structure units compared with the classical methyltransferases. Two loop regions undergo conformational changes upon AdoHcy binding. In contrast to the extended conformation of the cofactor seen in the classical methyltransferase structures, AdoHcy binds to HI0766 in a bent conformation. HI0766 and its close sequence relatives are all shorter versions of the more remotely related rRNA/tRNA methyltransferases of the spoU sequence family. We propose that the spoU sequence family contains the same core domain for cofactor binding as HI0766 but has an additional domain for substrate binding. The substrate-binding domain is absent in HI0766 sequence family and may be provided by another Haemophilus influenzae partner protein, which is yet to be identified.

A catalytic mechanism for D-Tyr-tRNATyr deacylase based on the crystal structure of Hemophilus influenzae HI0670.

D-Tyr-tRNA(Tyr) deacylase is an editing enzyme that removes d-tyrosine and other d-amino acids from charged tRNAs, thereby preventing incorrect incorporation of d-amino acids into proteins. A model for the catalytic mechanism of this enzyme is proposed based on the crystal structure of the enzyme from Haemophilus influenzae determined at a 1.64-A resolution. Structural comparison of this dimeric enzyme with the very similar structure of the enzyme from Escherichia coli together with sequence analyses indicate that the active site is located in the dimer interface within a depression that includes an invariant threonine residue, Thr-80. The active site contains an oxyanion hole formed by the main chain nitrogen atoms of Thr-80 and Phe-79 and the side chain amide group of the invariant Gln-78. The Michaelis complex between the enzyme and D-Tyr-tRNA was modeled assuming a nucleophilic attack on the carbonyl carbon of D-Tyr by the Thr-80 O(gamma) atom and a role for the oxyanion hole in stabilizing the negatively charged tetrahedral transition states. The model is consistent with all of the available data on substrate specificity. Based on this model, we propose a substrate-assisted acylation/deacylation-catalytic mechanism in which the amino group of the D-Tyr is deprotonated and serves as the general base.

The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: structure, sequence analyses, and solution studies.

The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 A. The molecules form an up-down, four-helix bundle, and associate into homodimers. The fold is most closely related to the substrate-binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide- and substrate-binding domains on the respective genomes. Both HI0073 and HI0074 were annotated "hypothetical" in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double- and single-stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer.

From structure to function: YrbI from Haemophilus influenzae (HI1679) is a phosphatase.

The crystal structure of the YrbI protein from Haemophilus influenzae (HI1679) was determined at a 1.67-A resolution. The function of the protein had not been assigned previously, and it is annotated as hypothetical in sequence databases. The protein exhibits the alpha/beta-hydrolase fold (also termed the Rossmann fold) and resembles most closely the fold of the L-2-haloacid dehalogenase (HAD) superfamily. Following this observation, a detailed sequence analysis revealed remote homology to two members of the HAD superfamily, the P-domain of Ca(2+) ATPase and phosphoserine phosphatase. The 19-kDa chains of HI1679 form a tetramer both in solution and in the crystalline form. The four monomers are arranged in a ring such that four beta-hairpin loops, each inserted after the first beta-strand of the core alpha/beta-fold, form an eight-stranded barrel at the center of the assembly. Four active sites are located at the subunit interfaces. Each active site is occupied by a cobalt ion, a metal used for crystallization. The cobalt is octahedrally coordinated to two aspartate side-chains, a backbone oxygen, and three solvent molecules, indicating that the physiological metal may be magnesium. HI1679 hydrolyzes a number of phosphates, including 6-phosphogluconate and phosphotyrosine, suggesting that it functions as a phosphatase in vivo. The physiological substrate is yet to be identified; however the location of the gene on the yrb operon suggests involvement in sugar metabolism.

Assisting functional assignment for hypothetical Heamophilus influenzae gene products through structural genomics.

The three-dimensional structures of Haemophilus influenzae proteins whose biological functions are unknown are being determined as part of a structural genomics project to ask whether structural information can assist in assigning the functions of proteins. The structures of the hypothetical proteins are being used to guide further studies and narrow the field of such studies for ultimately determining protein function. An outline of the structural genomics methodological approach is provided along with summaries of a number of completed and in progress crystallographic and NMR structure determinations. With more than twenty-five structures determined at this point and with many more in various stages of completion, the results are encouraging in that some level of functional understanding can be deduced from experimentally solved structures. In addition to aiding in functional assignment, this effort is identifying a number of possible new targets for drug development.