C-terminal Histidine Research Articles

Structure–activity studies of GmSubPep, a soybean peptide defense signal derived from an extracellular protease

GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1–11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4–12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.

The CXC Chemokine-degrading Protease SpyCep of Streptococcus pyogenes Promotes Its Uptake into Endothelial Cells

Streptococcus pyogenes expresses the LPXTG motif-containing cell envelope serine protease SpyCep (also called ScpC, PrtS) that degrades and inactivates the major chemoattractant interleukin 8 (IL-8), thereby impairing host neutrophil recruitment. In this study, we identified a novel function of SpyCep: the ability to mediate uptake into primary human endothelial cells. SpyCep triggered its uptake into endothelial cells but not into human epithelial cells originating from pharynx or lung, indicating an endothelial cell-specific uptake mechanism. SpyCep mediated cellular invasion by an endosomal/lysosomal pathway distinct from the caveolae-mediated invasion pathway of S. pyogenes. Recombinant expression and purification of proteolytically active SpyCep and a series of subfragments allowed functional dissection of the domains responsible for endothelial cell invasion and IL-8 degradation. The N-terminal PR domain was sufficient to mediate endothelial cell invasion, whereas for IL-8-degrading activity, the protease domain and the flanking A domain were required. A polyclonal rabbit serum raised against the recombinant protease efficiently blocked the invasion-mediating activity of SpyCep but not its proteolytic function, further indicating that SpyCep-mediated internalization is independent from its enzymatic activity. SpyCep may thus specifically mediate its own uptake as secreted protein into human endothelial cells.

Interactions between Ni 2+ ions and tetrapeptides containing (Asp/Lys)HisGly( l/ d)His sequence

A series of linear tetrapeptides containing two histidyl residues in positions 2 and 4 with different chirality: DHGH, DHG( d-His), KHGH, KHG( d-His), Ac-DHGH-NH 2, Ac-DHG( d-His)-NH 2, Ac-KHGH-NH 2, and Ac-KHG( d-His)-NH 2 were synthesized, characterized and their binding properties towards Ni 2+ were investigated. To establish the stoichiometry and the stability of the resulting Ni 2+ complexes, potentiometric titrations were carried out. The coordination mode of the complexes formed was investigated by performing extensive spectroscopic analyses (UV–Vis, CD) in strict correlation with the potentiometric results. The effects of the nature of the first amino acid (Lys versus Asp) and of the N-terminal amino group acetylation were determined. A careful comparison of the Ni 2+ coordination abilities of the linear peptides provides a specific insight into the impact of the chirality of the C-terminal histidine residue (His 4) on the metal binding properties.

Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins

hspA encodes a small heat shock protein (sHSP) in Xanthomonas campestris pv. campestris, the causative agent of black rot in cruciferous plants. In this study, two-dimensional gel electrophoresis, promoter activity assays, and Northern hybridization results revealed that HspA expression was induced by heat shock but not by other stresses, although low-level expression was detectable by reverse transcription-polymerase chain reaction (RT-PCR) under normal culture conditions. An hspA mutant exhibited reduced tolerance to heat, especially in the presence of MgSO4, but no change in pathogenicity. Results of size-exclusion chromatography indicated that purified HspA(his), containing six C-terminal histidine residues, formed two different size classes of oligomeric complexes--410 and 820 kDa. In contrast, HspA(ter), the unmodified protein translated from the original hspA gene, formed only the 820-kDa complex. These results suggest that the C-terminus of HspA is important for oligomerization. Both HspA820(his) and HspA410(his) were able to partially protect luciferase against heat-induced aggregation. Unlike other reported sHSPs that commonly capture denaturing proteins in refoldable states until refolded by adenosine triphosphate-dependent chaperone systems, HspA(his) alone was capable of reactivating heat-inactivated EcoRI. Thus, Xanthomonas campestris pv. campestris HspA has potential application as a protective agent during the storage of proteins.

Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture

Lysyl oxidase is a highly insoluble enzyme requiring high concentrations of urea to solubilize. A method to obtain lysyl oxidase in high yields directly from an Escherichia coli culture without the need for refolding of inclusion bodies has been developed using nutrient rich media. pET21b was used to overexpress the lysyl oxidase enzyme and to introduce a C-terminal 6X histidine tag for purification. Lysyl oxidase yields of 10 mg of active and properly folded enzyme per liter of media have been obtained. Purification was achieved via affinity chromatography using a Ni–NTA column. Copper content was found to be 19%. LTQ cofactor formation in LOX is a self-processing event in the presence of copper. LTQ content was determined to be 24% based on reaction with phenylhydrazine to form a phenylhydrazone adduct. Quantification of this adduct was attained using the previously reported extinction coefficient of 15.4 mM −1cm −1. LTQ presence was also verified by redox cycling. Specific enzymatic activity was measured to be 0.31 U/mg, one of the highest activities reported.

Abstract 2575: Activation of antibody drug conjugate MDX-1203 by human carboxylesterase

Abstract MDX-1203 is a human anti-CD70 antibody conjugated to a DNA alkylating cytotoxic Drug A. Drug A is an enzyme-activated prodrug that requires cleavage of its peptide linker and removal of an ester-linked protecting group for cytotoxic activity. Upon internalization of MDX-1203 in targeted cancer cells, peptide linker in drug A is cleaved by lysosomal enzymes to produce Drug B; which is further processed by intracellular enzymes to produce active drug. The purpose of this study was to determine if Drug B and n-acetyl-cysteine-conjugated Drug A (NAC-Drug A) are substrates of recombinant human carboxylesterase, and to determine kinetic parameters for the enzyme iso-forms rhCE1 and rhCE2. Furthermore, a comparison of rhCE1 and rhCE2 kinetics was made with CPT-11, a commercially available prodrug used to treat colon cancer that is preferentially activated by human CE2. Human carboxylesterase 1 and 2 with a C-terminal histidine tag were cloned and expressed in CHO cells. The enzymes were purified using immobilized metal ion affinity chromatography (IMAC), and their identities were confirmed by N-terminal protein sequencing and Western blot using an anti-his antibody. Enzyme reactions were carried out at 37°C by incubation with Drug A, NAC-Drug B, or CPT-11. The amount of enzymatically released drug product was determined by RP-HPLC. Drug B was determined to be a substrate for both rhCE1 and rhCE2; however, its Km was much lower with rhCE2 (1.5 uM) as compared to rhCE1 (53 uM), and the overall enzyme efficiency (Vmax/Km) was approximately 50 fold higher for rhCE2 as compared to rhCE1. Similarly, Drug B and NAC-Drug A were both found to be better substrates for rhCE2 as compared to rhCE1. Furthermore, recombinant human CE2 cleaves ester group in NAC-Drug A, 20 fold more efficiently as compared to CPT-11. These results indicate that expression of carboxylesterase 2 in targeted cancer cells is probably important for intracellular activation of MDX-1203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2575.

Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3

Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of approximately 90% at approximately 250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from approximately 30 min without mCAH3 to approximately 10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.

Purification and On-Column Activation of a Recombinant Histidine-Tagged Pro-Transglutaminase after Soluble Expression inEscherichia coli

Microbial transglutaminase (MTG) is widely used as a protein crosslinking enzyme. Pro-transglutaminase from Streptomyces mobaraensis was expressed in Escherichia coli as a fusion protein carrying a C-terminal histidine tag (pro-MTG-His(6)) under high-density culture. A new method of on-column activation was designed for production. According to SDS-PAGE, 88.9% of pro-MTG-His(6) was transferred to mature MTG-His(6) with storage stabilization.

Caenorhabditis elegans metallothionein isoform specificity – metal binding abilities and the role of histidine in CeMT1 and CeMT2

Two metallothionein (MT) isoforms have been identified in the model nematode Caenorhabditis elegans: CeMT1 and CeMT2, comprising two polypeptides that are 75 and 63 residues in length, respectively. Both isoforms encompass a conserved cysteine pattern (19 in CeMT1 and 18 in CeMT2) and, most significantly, as a result of their coordinative potential, CeMT1 includes four histidines, whereas CeMT2 has only one. In the present study, we present a comprehensive and comparative analysis of the metal [Zn(II), Cd(II) and Cu(I)] binding abilities of CeMT1 and CeMT2, performed through spectroscopic and spectrometric characterization of the recombinant metal-MT complexes synthesized for wild-type isoforms (CeMT1 and CeMT2), their separate N- and C-terminal moieties (NtCeMT1, CtCeMT1, NtCeMT2 and CtCeMT2) and a DeltaHisCeMT2 mutant. The corresponding in vitro Zn/Cd- and Zn/Cu-replacement and acidification/renaturalization processes have also been studied, as well as protein modification strategies that make it possible to identify and quantify the contribution of the histidine residues to metal coordination. Overall, the data obtained in the present study are consistent with a scenario where both isoforms exhibit a clear preference for divalent metal ion binding, rather than for Cu coordination, although this preference is more pronounced towards cadmium for CeMT2, whereas it is markedly clearer towards Zn for CeMT1. The presence of histidines in these MTs is revealed to be decisive for their coordination performance. In CeMT1, they contribute to the binding of a seventh Zn(II) ion in relation to the M(II)(6)-CeMT2 complexes, both when synthesized in the presence of supplemented Zn(II) or Cd(II). In CeMT2, the unique C-terminal histidine abolishes the Cu-thionein character that this isoform would otherwise exhibit.

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl(2) (2.1 A; R = 18.2%, R(free) = 21.9%), and in complex with glutamate, MgCl(2), and ADP (2.7 A; R = 19.0%, R(free) = 24.2%). Inspection of these structures reveals an unusual binding pocket for the alpha-carboxylate of the glutamate substrate and an ATP-independent Mg(2+) coordination site, clarifying the Mg(2+) dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

Binding site of C-reactive protein on M-ficolin

The binding abilities of human C-reactive protein (CRP) with the C-terminal fibrinogen-like (FBG) domain and the full-length form of human M-ficolin were investigated by pull-down and zonal affinity chromatography analyses. Pull-down assays using an N-acetyl-d-glucosamine (GlcNAc)-agarose column demonstrated that CRP binds to the trimeric FBG domains, and that the GlcNAc-binding ability of the FBG domain is unaffected by CRP binding. Interestingly, the full-length M-ficolin, comprising the N-terminal collagen-like (COL) and C-terminal FBG domains, displayed lower affinity for CRP, and the monomeric FBG domain showed virtually no binding to CRP, as qualitatively judged by zonal affinity chromatography using a GlcNAc column. These results indicated that CRP binding requires the trimeric form of the FBG domain, and that the presence of the COL domain reduces the interaction between CRP and M-ficolin. In addition, pull-down assays using a histidine-tag affinity column demonstrated that neither the full-length M-ficolin nor the trimeric FBG domains, immobilized through their C-terminal histidine tags, showed any affinity for CRP, indicating that the CRP binding site is located near Ala326 at the C-terminus of M-ficolin, spatially close to a neck region (around Pro115) between the FBG and COL domains. From these findings, we concluded that CRP binding is enhanced by conformational bending at the neck region of M-ficolin, to avoid steric hindrance by the COL domain. Such a situation may be generated by oligomeric M-ficolin binding to surfaces with widely distributed ligands, such as pathogens.

PEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants

Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV-HT) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved [Sainsbury, F. and Lomonossoff, G.P. (2008)Plant Physiol. 148, 1212-1218]. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of non-essential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.

The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini

Studies on Carboxypeptidase Digests of Human Hemoglobin (Antonini, E., Wyman, J., Zito, R., Rossi-Fanelli, A., and Caputo, A. (1961) J. Biol. Chem. 236, PC60–PC63) The Effect of Oxygenation on the Rate of Digestion of Human Hemoglobins by Carboxypeptidases (Zito, R., Antonini, E., and Wyman, J. (1964) J. Biol. Chem. 239, 1804–1808)

Non-AUG translational initiation of a short CAPC transcript generating protein isoform

CAPC (also known as LRRC26) is a new gene with restricted expression in normal tissues, and with expression in many cancers and cancer cell lines. We have identified and characterized a short-transcript of CAPC ( S-CAPC). The nucleotide sequence analysis of CAPC mRNA showed that the transcription for S-CAPC starts at position +610 on the L-CAPC transcript. Interestingly, no translation initiation codon ‘AUG’ is present in this transcript. To determine if a non-AUG start site is utilized, the S-CAPC sequence was cloned into an expression vector with C-terminal myc and histidine tags, and transfected into 293T cells. Western blot and MALDI-TOF MS analysis on purified S-CAPC gave two distinct peaks at approximately 7.5 kDa. N-terminal amino acid sequencing of the purified 7.5 kDa protein product indicated that translation starts at the codon for cysteine on the S-CAPC transcript generating a 7.5 kDa CAPC protein products translated from a non-AUG initiation site.

Conformational Exchange in Pseudoazurin: Different Kinds of Microsecond to Millisecond Dynamics Characterized by Their pH and Buffer Dependence Using 15N NMR Relaxation

The dynamics of the reduced form of the blue copper protein pseudoazurin from Alcaligenes faecalis S-6 was investigated using (15)N relaxation measurements with a focus on the dynamics of the micro- to millisecond time scale. Different types of conformational exchange processes are observed in the protein on this time scale. At low pH, the protonation of the C-terminal copper-ligated histidine, His81, is observed. A comparison of the exchange rates in the presence and absence of added buffers shows that the protonation is the rate-limiting step at low buffer concentrations. This finding agrees with previous observations for other blue copper proteins, e.g., amicyanin and plastocyanin. However, in contrast to plastocyanin but similar to amicyanin, a second conformational exchange between different conformations of the protonated copper site is observed at low pH, most likely triggered by the protonation of His81. This process has been further characterized using CPMG dispersion methods and is found to occur with a rate of a few thousands per second. Finally, micro- to millisecond motions are observed in one of the loop regions and in the alpha-helical regions. These motions are unaffected by pH and are unrelated to the conformational changes in the active site of pseudoazurin.

Structural analysis of the adenovirus type 2 E3/19K protein using mutagenesis and a panel of conformation-sensitive monoclonal antibodies

The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide–MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3–13, 15–21 and 41–45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six β-strands and an α-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition.

Expression, purification, and reconstitution of the Na+/H+ exchanger sod2 in Saccharomyces cerevisiae

Sod2, is a Na(+)/H(+) exchanger present on the cytoplasmic membrane of the fission yeast Schizosaccharomyces pombe. It expels toxic Na(+) from the cytosol. Sod2 was expressed in Saccharomyces cerevisiae with a C-terminal histidine tag under control of the GAL1 promoter. Western blots using anti-V5 antibodies identified the tagged protein. Solubilization of the protein was by n-dodecyl beta-D: -maltoside. Immobilized Ni-ion column affinity chromatography partially purified the protein at a yield of ~240 microg per liter of culture. Sod2 was present as a 40-kDa and an 80-kDa protein, however, it co-purified with a number of other proteins. Cross linking of sod2 with N,N'-(o-phenylene)dimaleimide showed that sod2 was present in association with a number of other proteins as a larger molecular weight complex. Partially purified sod2 protein was reconstituted in proteoliposomes and functionally active. Our results suggest that the sod2 protein associates with a number of other proteins and can be expressed in S. cerevisiae in active form.

An engineered folded PLP‐bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.

Substitution of Arg230 and Arg233 in Escherichia coli Elongation Factor Tu Strongly Enhances Its Pulvomycin Resistance

Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP). Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two mutants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin. Moreover, the mutant [R230V, R233F]EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.

Active site of mycobacterial dUTPase: Structural characteristics and a built-in sensor

dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, α,β-imido-dUTP and Mg 2+ at 1.5 Å resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K d for α,β-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.