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Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.

Aminoacyl-tRNA synthetases are an indispensable component of ribosomal protein translational machinery and Plasmodium Tyrosyl-tRNA synthetase (PfTyrRS) is a validated drug target. This manuscript illustrates the dynamic conformational landscape of PfTyrRS in the context of substrate binding. Molecular dynamics simulations of PfTyrRS in the presence and absence of ligand show conformational heterogeneity for both the protein and the bound ligand. Diverse conformations for the evolutionarily conserved ATP binding motif (KMSKS) have been observed in both apo- and holo PfTyrRS. Further, the presented attributes of the tyrosyl-adenylate conformational sub-states in situ along with their implications on the strength of intermolecular interactions would be a pertinent benchmark for molecular design studies. In addition, an analysis of the ligand hydration pattern foregrounds the structurally conserved water-mediated inter-molecular interactions. The quantitative assessment of the conformational landscape, based on the fluctuations of the distance between the ligand binding pockets, of apo-PfTyrRS and holo-PfTyrRS highlights the nature of diversity in conformational sampling for the two cases. Evidently, the holo-PfTyrRS adopts a rather compact conformation compared to the apo-PfTyrRS. An intriguing asymmetry in the dynamics of the two monomers is contextualized with the functional asymmetry of the symmetrically dimeric PfTyrRS. Importantly, the network of non-bonded contacts in the apo- and holo- simulated ensembles has been analyzed. The graph-theoretic analysis-based novel insights concerning the nature of information flow as a function of ligation state would prove valuable in understanding PfTyrRS functions. The results presented here contend that understanding allostery in PfTyrRS is essential to astutely design structure-based inhibitors.

Nitrate transport in cyanobacteria is mediated by ABC-transporter, which consists of a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). Under salt stress, recombinant glycinebetaine (GB) not only protected the rate of nitrate transport in transgenic Anabaena PCC 7120, rather stimulated the rate by interacting with the ABC-transporter proteins. In silico analyses revealed that nrtA protein consisted of 427 amino acids, the majority of which were hydrophobic and contained a Tat (twin-arginine translocation) signal profile of 34 amino acids (1–34). The nrtC subunit of 657 amino acids contained two hydrophobic distinct domains; the N-terminal (5–228 amino acids), which was 59% identical to nrtD (the ATP-binding subunit) and the C-terminal (268–591), 28.2% identical to nrtA, suggesting C-terminal as a solute binding domain and N-terminal as ATP binding domain. Subunit nrtD consisted of 277 amino acids and its N-terminal (21–254) was an ATP binding motif. Phylogenetic analysis revealed that nitrate-ABC-transporter proteins are highly conserved among the cyanobacterial species, though variation existed in sequences resulting in several subclades. Nostoc PCC 7120 was very close to Anabaena variabilis ATCC 29413, Anabaena sp. 4–3 and Anabaena sp. CA = ATCC 33047. On the other, Nostoc spp. NIES-3756 and PCC 7524 were often found in the same subclade suggesting more work before referring it to Anabaena PCC 7120 or Nostoc PCC 7120. The molecular interaction of nitrate with nrtA was hydrophilic, while hydrophobic with nrtC and nrtD. GB interaction with nrtACD was hydrophobic and showed higher affinity compared to nitrate.

FleN generally functions as an antagonist of FleQ in regulating flagellar genes and biofilm matrix related genes in Pseudomonas aeruginosa. Here, we found that in Pseudomonas putida KT2440, FleN and FleQ play a synergistic role in regulating two biofilm matrix coding operons, lapA and bcs. FleN deletion decreased the transcription of lapA and increased the transcription of bcs operon, and the same trend was observed in fleQ deletion mutant before. In vitro experiments showed that FleN promoted the binding of FleQ to the lapA/bcs promoter DNA especially in the presence of ATP. Both phenotype observation and transcription analysis showed that, similar to fleQ deletion, fleN deletion significantly weaken the effect of high c-di-GMP level on biofilm formation, surface winkle phenotype and expression of lapA and bcs operons. Mutagenesis of the putative ATP binding motif in FleNK21Q revealed that FleN ATPase activity played an essential role in the regulation of flagellar number and swimming motility but was not critical for biofilm formation. Our results revealed that FleN was not an antagonist of FleQ but a synergistic factor of FleQ in regulating the two biofilm matrix coding operons in P. putida KT2440.

Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc.

S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C bungeana S-adenosylmethionine synthetase), from C bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919 bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C bungeana. (C) 2015 Elsevier B.V. All rights reserved.

In many organisms ``Universal Stress Proteins'' CUSPS) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8 angstrom and 2.4 angstrom resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800 daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing. (C) 2015 Elsevier Inc. All rights reserved.

Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in thiazole/oxazole-modified microcin (TOMM) natural products remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 ubiquitin-activating enzymes, whereas the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif. This motif is conserved among TOMM YcaOs and is required for cyclodehydration. Furthermore, we demonstrate that the C protein regulates substrate binding and catalysis and that the proline-rich C terminus of the D protein is involved in C protein recognition and catalysis. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.

The aspS gene encoding Aspartyl-tRNA synthetase (AspRS) from a thermotolerant acetic acid bacterium, Acetobacter pasteurianus SKU1108, has been cloned and characterized. The open reading frame (ORF) of the aspS gene consists of 1,788 bp, encoding 595 amino acid residues. The highly conserved Gly-Val-Asp-Arg ATP binding motif (motif 3) is located at the position 537-540 in the C-terminus. Deletion analysis of the aspS gene upstream region suggested that the promoter is around 173 bp upstream from the ATG initiation codon. Interestingly, transformation with the plasmids pGEM-T138, pUC138, and pCM138 synthesizing 138 amino acid C-terminal fragments of AspRS, that carry the ATP binding domain, caused E. coli cell lengthening at 37 and 42°C. Moreover, E. coli harboring pUC595 (synthesizing all 595 amino acids) and a disordered aspS gene in pGEM-T138 had normal rod shapes. The normal rod shape was observed in E. coli harboring pD539V following site-directed mutagenesis of the ATP binding domain. We propose that over-production of truncated C-terminal peptides of AspRS may cause sequestration of intracellular ATP in E. coli, leaving less ATP for cell division or shaping cell morphology.

An Universal Stress Protein (USP) expressed under acid stress condition by Listeria innocua ATCC 33090 was investigated. The USP was up-regulated not only in the stationary phase but also during the exponential growth phase. The three dimensional (3D) structure of USP was predicted using a combined proteomic and bioinformatics approach. Phylogenetic analysis showed that the USP from Listeria detected in our study was distant from the USPs of other bacteria (such as Pseudomonas spp., Escherichia coli, Salmonella spp.) and clustered in a separate and heterogeneous class including several USPs from Listeria spp. and Lactobacillus spp. An important information on the studied USP was obtained from the 3D-structure established through the homology modeling procedure. In detail, the Model_USP-691 suggested that the investigated USP had a homo-tetrameric quaternary structure. Each monomer presented an architecture analogous to the Rossmann-like α/β-fold with five parallel β-strands, and four a-helices. The analysis of monomer-monomer interfaces and quality of the structure alignments confirmed the model reliability. In fact, the structurally and sequentially conserved hydrophobic residues of the β-strand 5 (in particular the residues V146 and V148) were involved in the inter-chains contact. Moreover, the highly conserved residues I139 and H141 in the region α4 were involved in the dimer association and functioned as hot spots into monomer-monomer interface assembly. The hypothetical assembly of dimers was also supported by the large interface area and by the negative value of solvation free energy gain upon interface interaction. Finally, the structurally conserved ATP-binding motif G-2X-G-9X-G(S/T-N) suggested for a putative role of ATP in stabilizing the tetrameric assembly of the USP. Therefore, the results obtained from a multiple approach, consisting in the application of kinetic, proteomic, phylogenetic and modeling analyses, suggest that Listeria USP could be considered a new type of ATP-binding USP involved in the response to acid stress condition during the exponential growth phase.

An Universal Stress Protein (USP) expressed under acid stress condition by Listeria innocua ATCC 33090 was investigated. The USP was up-regulated not only in the stationary phase but also during the exponential growth phase. The three dimensional (3D) structure of USP was predicted using a combined proteomic and bioinformatics approach. Phylogenetic analysis showed that the USP from Listeria detected in our study was distant from the USPs of other bacteria (such as Pseudomonas spp., Escherichia coli, Salmonella spp.) and clustered in a separate and heterogeneous class including several USPs from Listeria spp. and Lactobacillus spp. An important information on the studied USP was obtained from the 3D-structure established through the homology modeling procedure. In detail, the Model_USP-691 suggested that the investigated USP had a homo-tetrameric quaternary structure. Each monomer presented an architecture analogous to the Rossmann-like α/β-fold with five parallel β-strands, and four α-helices. The analysis of monomer-monomer interfaces and quality of the structure alignments confirmed the model reliability. In fact, the structurally and sequentially conserved hydrophobic residues of the β-strand 5 (in particular the residues V146 and V148) were involved in the inter-chains contact. Moreover, the highly conserved residues I139 and H141 in the region α4 were involved in the dimer association and functioned as hot spots into monomer–monomer interface assembly. The hypothetical assembly of dimers was also supported by the large interface area and by the negative value of solvation free energy gain upon interface interaction. Finally, the structurally conserved ATP-binding motif G-2X-G-9X-G(S/T-N) suggested for a putative role of ATP in stabilizing the tetrameric assembly of the USP. Therefore, the results obtained from a multiple approach, consisting in the application of kinetic, proteomic, phylogenetic and modeling analyses, suggest that Listeria USP could be considered a new type of ATP-binding USP involved in the response to acid stress condition during the exponential growth phase.

Voltage-dependent anion channel (VDAC) proteins abound in the outer membrane of mitochondria. They play an important role in mitochondrial membrane permeabilization (MMP), which can lead to stress-induced cellular apoptosis and necrosis. Several pathogens regulate this MMP in their host cells to benefit their replication cycle, while in other cases, the host can use the same mechanism to combat pathogenesis. In this study, the first shrimp VDAC gene was identified and characterized from Marsupenaeus japonicus (MjVDAC). Its open reading frame (ORF) contained 849 bp encoding 282 amino acids. The deduced MjVDAC protein includes the 4-element eukaryotic porin signature motif, the conserved ATP binding motif (the GLK motif) and a VKAKV-like sequence known in other organisms to be involved in the protein's incorporation in the mitochondrial outer membrane. Tissue tropism analysis indicated that MjVDAC is abundant in the heart, muscle, stomach and pleopod. MjVDAC proteins colocalized with mitochondria in transiently transfected Sf9 cells and in shrimp hemocytes. dsRNA silencing of shrimp VDAC delayed white spot syndrome virus (WSSV) infection by 1 day in different shrimp organs. Taken together, these findings suggest that MjVDAC is likely to be involved in WSSV pathogenesis.

The structure of the nucleotide-binding domain of the Mg-ATPase MgtA from Escherichia coli has been solved and refined to 1.6 A resolution. The structure is made up of a six-stranded beta-sheet and a bundle of three alpha-helices, with the nucleotide-binding site sandwiched in between. The MgtA nucleotide-binding domain is shorter and more compact compared with that of the related Ca-ATPase and lacks one of the beta-strands at the edge of the beta-sheet. The ATP-binding pocket is surrounded by three sequence and structural motifs known from other P-type ATPases and a fourth unique motif that is found only in Mg-ATPases. This motif consists of a short polypeptide stretch running very close to the ATP-binding site, while in Ca-ATPase the binding site is more open, with the corresponding polypeptide segment folded away from the active site.

The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-Å crystal structure of Buk2 complexed with (β,γ-methylene) adenosine 5′-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

In an effort to use monoclonal antibodies (mAbs) as selective probes for early detection of breast cancer, the specificities of a number of antipeptide mAbs have been studied at the individual amino acid level using single substitution peptide analogs and peptide combinatorial libraries. In this study, the mapping results are presented for mAbl72-12A4, which was raised against the haptenic peptide LGSGAFGTIYKG(C), corresponding to residues 138–149 of the oncogene v-erbB. This peptide is homologous with a region in epidermal growth factor receptor (EGFR) and human oncogene c-erbB-2, and contains the ATP binding motif that is common among protein kinases. The substitution profile of this interaction correlated well with the results from the screening of hexa- and decapeptide positional scanning libraries. Based on the results of this mAb's specificity for the antigenic determinant (-AFGTIYK-), proteins that have sequence homology were found from a database search of human sequences. Thirty-two unique peptide sequences, a majority of which was from protein kinases, were synthesized and tested for recognition by mAb 172–12A4. Eleven peptides had activities that differed from the original peptide by less than an order of magnitude, and the activities for 29 of the 32 (90%) could be accurately predicted based on the individual substitution analog results. While both epitope mapping approaches address the amino acid level of mAb specificity, positional scanning libraries offer an advantage of identifying the positional importance of each antigenic determinant residue without any prior knowledge of the mAb's specificity. The fine specificity mapping of peptide-specific mAbs using the synthetic tools illustrated here will be useful for the development of immunodiagnostics that detect cancer-related proteins in clinical samples.

Genomic and transcriptomic studies have revealed a sophisticated and powerful apoptosis regulation network in oyster, highlighting its adaptation to sessile life in a highly stressful intertidal environment. However, the functional molecular basis of apoptosis remains largely unexplored in oysters. In this study, we focused on a representative apoptotic gene encoding voltage-dependent anion channel 2 (VDAC2), a porin that abounds at the mitochondrial outer membrane. This is the first report on the identification and characterization of a VDAC gene in the Pacific oyster, Crassostrea gigas (CgVDAC2). The full length of CgVDAC2 was 1,738 bp with an open reading frame of 843 bp that encoded a protein of 281 amino acids. A four-element eukaryotic porin signature motif, a conserved ATP binding motif, and a VKAKV-like sequence were identified in the predicted CgVDAC2. Expression pattern analysis in different tissues and developmental stages as well as upon infection by ostreid herpesvirus 1 revealed the energy supply-related and immunity-related expression of CgVDAC2. CgVDAC2 was co-localized with mitochondria when it was transiently transfected into HeLa cells. Overexpression of CgVDAC2 in HEK293T cells suppressed the UV irradiation-induced apoptosis by inhibiting the pro-apoptotic function of CgBak. RNA interference induced reduction in CgVDAC2 expression showed a promoted apoptosis level upon UV light irradiation in hemocytes. The yeast two-hybrid system and co-immunoprecipitation assay indicated a direct interaction between CgVDAC2 and the pro-apoptotic protein CgBak. This study revealed the function of VDAC2 in oyster and provided new insights into its involvement in apoptosis modulation and host defense in mollusks.

The three-dimensional crystal structure of the PduO-type corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) has been solved to 1.68-Å resolution. The functional assignment of LrPduO as a corrinoid adenosyltransferase was confirmed by in vivo and in vitro evidence. The enzyme has an apparent [Formula: see text] of 2.2 μM and [Formula: see text] of 0.13 μM and a k(cat) of 0.025 s(−1). Co-crystallization of the enzyme with Mg-ATP resulted in well-defined electron density for an N-terminal loop that had been disordered in other PduO-type enzyme structures. This newly defined N-terminal loop makes up the lower portion of the enzyme active site with the other half being contributed from an adjacent subunit. These results provide the first detailed description of the enzyme active site for a PduO-type adenosyltransferase and identify a unique ATP binding motif at the protein N terminus. The molecular architecture at the active site offers valuable new insight into the role of various residues responsible for the human disease methylmalonic aciduria.

Sporulation is an ancient developmental process that involves the formation of a highly resistant endospore within a larger mother cell. In the model organism Bacillus subtilis, sporulation-specific sigma factors activate compartment-specific transcriptional programs that drive spore morphogenesis. σG activity in the forespore depends on the formation of a secretion complex, known as the “feeding tube,” that bridges the mother cell and forespore and maintains forespore integrity. Even though these channel components are conserved in all spore formers, recent studies in the major nosocomial pathogen Clostridium difficile suggested that these components are dispensable for σG activity. In this study, we investigated the requirements of the SpoIIQ and SpoIIIA proteins during C. difficile sporulation. C. difficile spoIIQ, spoIIIA, and spoIIIAH mutants exhibited defects in engulfment, tethering of coat to the forespore, and heat-resistant spore formation, even though they activate σG at wildtype levels. Although the spoIIQ, spoIIIA, and spoIIIAH mutants were defective in engulfment, metabolic labeling studies revealed that they nevertheless actively transformed the peptidoglycan at the leading edge of engulfment. In vitro pull-down assays further demonstrated that C. difficile SpoIIQ directly interacts with SpoIIIAH. Interestingly, mutation of the conserved Walker A ATP binding motif, but not the Walker B ATP hydrolysis motif, disrupted SpoIIIAA function during C. difficile spore formation. This finding contrasts with B. subtilis, which requires both Walker A and B motifs for SpoIIIAA function. Taken together, our findings suggest that inhibiting SpoIIQ, SpoIIIAA, or SpoIIIAH function could prevent the formation of infectious C. difficile spores and thus disease transmission., Author Summary The bacterial spore-forming pathogen Clostridium difficile is a leading cause of nosocomial infections in the United States and represents a significant threat to healthcare systems around the world. As an obligate anaerobe, C. difficile must form spores in order to survive exit from the gastrointestinal tract. Accordingly, spore formation is essential for C. difficile disease transmission. Since the mechanisms controlling this process remain poorly characterized, we analyzed the importance of highly conserved secretion channel components during C. difficile sporulation. In the model organism Bacillus subtilis, this channel had previously been shown to function as a “feeding tube” that allows the mother cell to nurture the developing forespore and sustain transcription in the forespore. We show here that conserved components of this structure in C. difficile are dispensable for forespore transcription, although they are important for completing forespore engulfment and retaining the protective spore coat around the forespore, in contrast with B. subtilis. The results of our study suggest that targeting these conserved proteins could prevent C. difficile spore formation and thus disease transmission.

Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.

Two cDNA clones ( ZmFRK1 and ZmFRK2 ) encoding fructokinases (FRKs, E.C. 2.7.1.4) were isolated from maize ( Zea mays L.). They encode polypeptides of 323 and 335 amino acids, respectively. Both of the deduced proteins contain ATP binding motif and putative substrate recognition sequences. They share 73.7% sequence identity, and have high sequence homology with previously isolated FRKs from potato, sugar beet, tomato, and Arabidopsis . The in vitro enzymatic studies of the recombinant ZmFrk1 and ZmFrk2 proteins showed that they both had fructose specific kinase activities, but ZmFrk1 had much higher turnover rate than ZmFrk2. Both ZmFrk1 and ZmFrk2 were inhibited by the substrate fructose although ZmFrk1 was less sensitive. The mRNAs of both ZmFRK1 and ZmFRK2 accumulated to high levels in roots and stems, but were barely detectable in leaves. The expression pattern of the two genes was different during seed development. The transcript of ZmFRK1 only appeared in later stages of development, whereas the ZmFRK2 mRNA was expressed in earlier time. Taken together, our results imply that ZmFRK1 and ZmFRK2 play distinct biological roles during seed development.

Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells. Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown. Here, we identified a novel yeast gene, RSB1 (resistance to sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant. RSB1encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa. Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains. We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release. This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity. Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds. These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane.

Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In λ, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts., Author Summary Mycobacterium leprae is the causative organism of the disease so called leprosy. It posses global health challenge due to the emergence of drug resistant M. leprae bacilli. In search for new drug targeting strategies, study of bacterial energy metabolism is significant. Reports claim that M. leprae generates ATP, an important energy metabolite, which is very much required for its own biochemical activities and unlike other organisms, it needn’t require host species as a source of ATP. The growth and survival of these pathogenic bacteria in infected hosts are supported by an immunodominant antigenic membrane protein called HSP18. Hereby we report for the first time evidences of HSP18 and ATP interaction in vitro and quantified the same. We also identified the Walker-B ATP binding motif in M. leprae HSP18. Our study reveals that HSP18 when interacts with ATP, undergoes an enhancement in its anti-aggregation property. We demonstrate here a novel report on ability of HSP18 to prevent thermal inactivation of enzymes which is also enhanced in presence of ATP. Our study provides new insights into the effect of ATP on the structure and chaperone function of M. leprae HSP18 which may be critical for survival of M. leprae bacilli. This may provide a new basis for the development of new drug targeting strategy such as ATP competitive antibiotics/inhibitors in context of effective treatment of leprosy.

The six MCM (minichromosome maintenance) proteins are essential DNA replication factors that each contain a putative ATP binding motif and together form a heterohexameric complex. We show that these motifs are required for viability in vivo and coordinated ATP hydrolysis in vitro. Mutational analysis discriminates between two functionally distinct MCM protein subgroups: Mcm4p, 6p, and 7p contribute canonical ATP binding motifs essential for catalysis, whereas the related motifs in Mcm2p, 3p, and 5p serve a regulatory function. Reconstitution experiments indicate that specific functional interactions between these two subgroups are required for robust ATP hydrolysis. Our observations show parallels between the MCM complex and the F1-ATPase, and we discuss how ATP hydrolysis by the MCM complex might be coupled to DNA strand separation.

Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes inKm(ATP). Km for mevalonate increases by ≈20-fold for S146A, ≈40-fold for T243A, and 100-fold for S201A. Vmax changes for S145A, S201A, and T243A are ≤3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. Vmax for S146A is diminished by 4000-fold. In terms of V/KMVA, this substitution produces a 105-fold effect, suggesting an active site location and catalytic role for Ser-146. The largekcat effect suggests that Ser-146 productively orients ATP during catalysis. KD(Mg-ATP)increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg2+-ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.

CC3 is a metastasis suppressor that inhibits metastasis of the variant small cell lung carcinoma (v-SCLC) by predisposing cells to apoptosis. The same protein was also reported as a cellular cofactor, TIP30, which stimulates HIV-1 Tat-activated transcription by interacting with both Tat and RNA polymerase II. We report here that TIP30/CC3 is a novel serine/threonine kinase. It phosphorylates the heptapeptide repeats of the C-terminal domain (CTD) of the largest RNA polymerase II subunit in a Tat-dependent manner. Amino acid substitutions in the putative ATP binding motif that abolish the TIP30 kinase activity also inhibit the ability of TIP30 to enhance Tat-activated transcription or to sensitize NIH 3T3 and v-SCLC cells to apoptosis. Furthermore, ectopic expression of TIP30/CC3 in v–SCLC cells induces expression of a number of genes that include the apoptosis-related genes Bad and Siva, as well as metastasis suppressor NM23-H2. These data demonstrate a molecular mechanism for TIP30/CC3 function and suggest a novel pathway for regulating apoptosis.

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.

The P-loop is a common motif found in ATP- and GTP-binding proteins. The recently cloned murine ATP-sulfurylase/adenosine 5'-phosphosulfate (APS) kinase contains a P-loop (residues 59-66) in the APS kinase portion of the bifunctional protein. A series of enzymatic assays covering the multiplicity of functions of this unique protein (reverse ATP-sulfurylase, APS kinase, and an overall assay) were used to determine the effect of deleting or altering specific residues constituting this motif. In addition to the full-length cDNA construct (1MSK), two deletion mutants that progressively shortened the N terminus by 34 amino acids (2MSK) and 70 amino acids (3MSK) were designed to examine the effects of translation initiation before (2MSK) and after (3MSK) the P-loop. The 2MSK protein possessed sulfurylase and kinase activity equivalent to the full-length construct, but 3MSK exhibited no kinase activity and reduced sulfurylase activity. In light of the evident importance of this motif, a number of site-directed mutants were designed to investigate the contribution of key residues. Mutation of a highly conserved lysine in the P-loop to alanine (K65A) or arginine (K65R) or the following threonine (T66A) to alanine ablated APS kinase activity while leaving ATP-sulfurylase activity intact. Three mutations (G59A, G62A, and G64A) addressed the role of the conserved glycines as follows: G64A showed diminished APS kinase activity only, whereas G62A had no effect on either activity. G59A caused a significant decrease in ATP-sulfurylase activity without effect on APS kinase activity. A series of highly conserved flanking cysteines (Cys-53, Cys-77, and Cys-83) were mutated to alanine, but none of these mutations showed any effect on either enzyme activity.

The herpes simplex virus (HSV-1) UL15 gene encodes one of the six viral gene products required for viral DNA cleavage and packaging. UL15 is a spliced gene and encodes two separately translated proteins, UL15 and UL15.5. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase, a protein which cleaves the polymeric T4 DNA into monomers. Both proteins contain a putative ATP binding motif known as the Walker A and B boxes. In this report, immunofluorescence was used to show that UL15 localizes to the nucleus in the absence of any other viral proteins; this indicates that UL15 contains its own nuclear localization signal. In addition, we found that UL15 colocalizes with replication compartments at early times (6 h postinfection). Since, at this time, preformed capsids as well as other cleavage and packaging proteins are also recruited to replication compartments, it seems likely that cleavage and packaging occurs in the same compartments in which DNA synthesis occurs. Also in this report, we have investigated UL15.5, the N-terminally truncated gene product of the UL15 open reading frame (ORF). The start codon has been mapped to Met 443 within the UL15 ORF. Furthermore, we have shown that plasmids containing a UL15.5 knockout mutation still complement the growth of UL15 insertion mutant viruses, indicating that UL15.5 is not required for viral growth in cell culture. Last, we constructed a UL15 mutant, UL15C(G263A), in which the invariant Gly 263 in the Walker box A of the ATP binding motif (GKT) was substituted with an alanine. We show that the mutant gene fails to support the growth of UL15 insertion mutant viruses, indicating that the putative ATP binding motif of UL15 is indispensable for its function.

GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria. PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.

Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly.

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.

A cDNA encoding for a weakly inward rectifying K+ channel (sWIRK: salmon weakly inward rectifying K+ channel) was isolated from the masu salmon brain by expression cloning. The sWIRK channel exhibited the highest similarity with members of the ROMK1 subfamily, BIR10/KAB-2 (70% amino acid identity) and ROMK1 (46%). An ATP binding motif which is characteristic of this subfamily was also conserved. The sWIRK RNA was detected in the brain, but not in the heart, kidney, skeletal muscle, liver, testis, and ovary. In the brain, the expression was observed in the ependymoglial cells on the surface of the ventricles as well as in the small perineuronal glia-like cells in the midbrain and the medulla. When compared with the strong inward rectifier IRK1 channel, the sWIRK channel showed a much weaker inward rectification property, and the activation kinetics upon hyperpolarization was slower and less voltage-dependent. The slope conductance of the single channel inward current was 37 pS (140 mM K+o), and outward current channel events were also observed. The weak rectification of sWIRK is significant in that it has a negatively charged residue (glutamate) in the M2 region which is reported to cause strong inward rectification. By introducing a point mutation to remove this negative charge (glutamine), the sWIRK E179Q mutant channel lost its inward rectification property completely, and the single channel property (45 pS; 140 mM K+o) was ohmic up to highly depolarized potential, even in the presence of the physiological cytoplasmic blockers such as Mg2+ and polyamines.

Transcript levels from the P5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by the AAV Rep78 and Rep68 proteins in the absence of helper virus. We have identified a Rep-responsive negative cis element of the P5 promoter between the P5 TATA box and transcription start site by using 5' and 3' deletions of the P5 promoter fused to the chloramphenicol acetyltransferase gene. This element contains four imperfect GAGC repeats similar to the Rep recognition sequences (RRSs) in the AAV inverted terminal repeats and in the AAV preferred integration locus in chromosome 19. Band shift analyses showed that human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, formed Rep-specific complexes with this P5 RRS DNA. Within the P5 RRS, mutation of a cytosine at position 273 in the AAV sequence to guanine abolished Rep68 binding to the DNA. A mutation in the P5 RRS within a full-length AAV genome, which abolished Rep binding, resulted in a 40 to 50% reduction in the ability of wild-type Rep68 to inhibit the accumulation of P5 transcripts in vivo. In contrast, the Rep68/K340H mutant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negative regulation of P5 transcript levels by Rep68; one involves Rep68 binding to the P5 RRS, and another requires the region of Rep68 containing the consensus NTP-binding motif. Furthermore, our studies of AAV genomes containing mutated RRS- and/or YY1-binding elements suggest that transcription factor YY1 binding to the transcription start site of P5 interferes with Rep68 repression of the P5 promoter.

The nucleotide sequence of the amidase operon of Pseudomonas aeruginosa has been completed and two new genes identified amiB and amiS. The complete gene order for the operon is thus amiEBCRS. The amiB gene encodes a 42-kDa protein containing an ATP binding motif that shares extensive homology with the Clp family of proteins and also to an open reading frame adjacent to the amidase gene from Rhodococcus erythropolis. Deletion of the amiB gene has no apparent effect on inducible amidase expression and it is thus unlikely to encode a regulatory protein. A maltose-binding protein-AmiB fusion has been purified and shown to have an intrinsic ATPase activity (Km = 174 +/- 15 mM; Vmax = 2.4 +/- 0.1 mM/min/mg), which is effectively inhibited by ammonium vanadate and ADP. The amiS gene encodes an 18-kDa protein with a high content of hydrophobic residues. Hydropathy analysis suggests the presence of six transmembrane helices in this protein. The AmiS sequences is homologous to an open reading frame identified adjacent to the amidase gene from Mycobacterium smegmatis and to the ureI gene from the urease operon of Helicobacter pylori. AmiS and its homologs appear to be a novel family of integral membrane proteins. Together AmiB and AmiS resemble two components of an ABC transporter system.

The cbbL and cbbS genes, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), were cloned and sequenced from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The cbbL gene encoded a 474-amino-acid (aa) protein (53 285 Da); cbbS encoded a 124-aa protein (14656 Da). An ORF found downstream from the cbbLS genes encoded a 267-aa protein (29 565 Da) which had no similarity to cbbX located downstream from cbbLS from Alcaligenes eutrophus and Xanthobacter flavus . This gene, called cbbQ , was highly similar to the nirQ gene of the denitrification gene cluster from P. aeruginosa and P. stutzeri .

The ANFA protein is the transcriptional activator of the sigma 54-dependent anfHDGK operon, which codes for the structural genes of the third nitrogenase system in Azotobacter vinelandii. We have purified, in soluble active form, an N-terminally truncated form of the protein, delta ANFA, which activates transcription from the anfH promoter and other sigma 54-dependent promoters in a purified transcription system. Sequences upstream of the anfH promoter and the presence of the integration host factor protein stimulate transcription, and we have shown that delta ANFA binds to sites situated between 200 and 300 base pairs upstream of the anfH promoter. In common with other sigma 54-dependent activators, ANFA has a highly conserved ATP binding motif in its central domain, and we have demonstrated that ATP or GTP is required for productive complex formation and that the purified truncated protein has a constitutive ATPase activity, which is presumably required to drive open complex formation.

Vaccinia virus early transcription factor (VETF) activates the transcription of early gene templates by the viral RNA polymerase. VETF is a heterodimeric protein that binds to transcription promoters and has an associated DNA-dependent ATPase activity. The small subunit of VETF has sequences resembling two motifs commonly found in ATPases: an A-type ATP binding motif and a DEAH box. To investigate the functional role of the ATPase activity, we have analyzed the effect of mutations in each of the putative ATPase motifs. Recombinant VETF was expressed in HeLa cells using a vaccinia virus/T7 RNA polymerase system. Simultaneous expression of both subunits of VETF was required to obtain soluble protein with promoter binding, DNA-dependent ATPase, and transcription activation functions. The mutants with altered ATPase motifs retained promoter binding activity but had no detectable ATPase activity and no ability to activate transcription. The DEAH box mutant was shown to dominantly repress transcription activation by wildtype VETF. These results indicate that the DNA-dependent ATPase activity of VETF is essential for its transcription activation function.

A superfamily of ATPases is described with its members sharing three distinct conserved amino acid sequence motifs. The superfamily includes numerous proteins involved in active partitioning of bacterial plasmids and chromosomes, nitrogenase iron proteins (nifH gene products), the anion pump ATPase ArsA, and VirC1 proteins encoded by Agrobacterium Ti plasmids and apparently involved in formation of single-stranded plasmid DNA. A database search identified partial sequences of genes encoding putative human and archaebacterial chromosome partitioning ATPases, suggesting that these proteins fulfil a universal function in cell division. The proteins belonging to this superfamily show the transition from the classical fingerprint of the A type purine NTP-binding motif, GXXGXGK[ST], to a significantly modified signature, KGGXXK[ST], with the apparent preservation of the loop conformation typical of this motif. It is speculated that the ancestral form of the A motif might have comprised a loop rich in Gly residues, GXGGXGK[ST], resembling that in NifH proteins and ArsA. Some of the Gly residues might have been differentially substituted in various evolutionary lineages of NTPases. The functional diversity of the proteins of this ATPase superfamily is comparable with the range of functions described previously for the superfamily of "UvrA-related" ATPases.

The KMSKS motif is the ATP binding motif for aminoacylation process of class I aminoacyl-tRNA synthetases. Although researches based on natural proteins inform us about the contribution of natural amino acid sequences for the catalysis, they have difficulties in discussing the other alternative sequences and prohibited sequences for the motif to maintain the catalytic ability. In order to reveal such the conditions for the alternative and prohibited sequences, it is important to investigate a library of various mutants for the motif. For that purpose, we build a library of more than 200 mutants substituting the KMSSS loop, Lys204-Met205-Ser206-Ser207-Ser208, in tyrosyl-tRNA synthetase of Methanococcus jannaschii, and their catalytic abilities were examined by the Amber suppression method. Mutants of K204R and K204N still maintained catalytic abilities to a certain extent. On the other hand, a variety of alternative sequences for Ser206-Ser207-Ser208 were obtained, and some of those did not include either Ser or Thr, which were regarded as necessary residues in the KMSKS motif in previous works. In this article, catalytic activity of all the mutants are represented in detail and some suggestions for the condition of the motif are discussed.

A fundamental question in protein chemistry is how the native energy landscape of enzymes enables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studies have revealed that substrate binding is accompanied by large rate-limiting spatial displacements of both the ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe the native energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and in the presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATP binding motif populates both the open and the closed conformations with almost equal populations. A similar scenario is observed for AMP binding, which induces an equilibrium between open and closed conformations of the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes that exist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substrate binding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic coupling between the ATP and AMP binding sites was discovered. On the basis of these and previous results, we propose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existing equilibria toward catalytically active states.

p21-Activated kinases (PAKs) are regulators of cell motility and proliferation. PAK activity is regulated in part by phosphoinositide-dependent kinase 1 (PDK1). We hypothesized that reduced PAK activity was involved in the effects of 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide (OSU-03012), a previously characterized PDK1 inhibitor derived from celecoxib. In three human thyroid cancer cell lines, OSU-03012 inhibited cell proliferation with reduced AKT phosphorylation by PDK1. OSU-03012 unexpectedly inhibited PAK phosphorylation at lower concentrations than PDK1-dependent AKT phosphorylation in two of the three lines. In cell-free kinase assays, OSU-03012 was shown to inhibit PAK activity and compete with ATP binding. In addition, computer modeling predicted a docking site for OSU-03012 in the ATP binding motif of PAK1. Finally, overexpression of constitutively activated PAK1 partially rescued the ability of motile NPA thyroid cancer cells to migrate during OSU-03012 treatment, suggesting that inhibition of PAK may be involved in the cellular effects of OSU-03012 in these cells. In summary, OSU-03012 is a direct inhibitor of PAK, and inhibition of PAK, either directly or indirectly, may be involved in its biological effects in vitro.

The ε subunit of bacterial and chloroplast F o F 1 -ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound ε subunit from a thermophilic Bacillus PS3 at 1.9-Å resolution. The C-terminal two α-helices were folded into a hairpin, sitting on the β sandwich structure, as reported for Escherichia coli . A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli ε subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 ε subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the ε C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F o F 1 -ATP synthase.