Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.     

Aspartyl tRNA-synthetase from Escherichia coli: flexibility and adaptability to the substrates

The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A. The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP. The asymmetric unit contains three monomers of the enzyme. While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed. Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes. The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements. In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein.     

SIVcpz from a naturally infected Cameroonian chimpanzee: biological and genetic comparison with HIV-1 N

Thus far, simian immunodeficiency virus from chimpanzees (SIVcpz) genomes have been characterized as Pan troglodytes troglodytes and show a strong relation with human immunodeficiency virus (HIV)-1 N in their env genes. We fully characterized another SIVcpz from P. t. troglodytes. This chimpanzee (Cam5) was, as was also the host of SIVcpz-cam3, wild born in Cameroon, a region where all three groups of HIV-1 (M, N and O) co-occur. In contrast to other SIVcpz, SIVcpz-cam5 was isolated immediately after the rescue of the animal. Our data demonstrate that SIVcpz-cam5, like SIVcpz-cam3, grows easily on human peripheral blood mononuclear cells (PBMCs) and uses CCR5 as a co-receptor similar to HIV-1 N YBF30. Phylogenetic analysis based on the entire env gene shows that SIVcpz-cam5 falls into the same unique subcluster as HIV-1 N YBF30, SIVcpz-cam3 and SIVcpz-US. A phylogenetic relationship was also found with the vif gene of HIV-1 N. This study provides proof that HIV-1 N related viruses circulate in wild P. t. troglodytes.     

env sequences of simian immunodeficiency viruses from chimpanzees in Cameroon are strongly related to those of human immunodeficiency virus group N from the same geographic area

Human immunodeficiency virus type 1 (HIV-1) group N from Cameroon is phylogenetically close, in env, to the simian immunodeficiency virus (SIV) cpz-gab from Gabon and SIVcpz-US of unknown geographic origin. We screened 29 wild-born Cameroonian chimpanzees and found that three (Cam3, Cam4, and Cam5) were positive for HIV-1 by Western blotting. Mitochondrial DNA sequence analysis demonstrated that Cam3 and Cam5 belonged to Pan troglodytes troglodytes and that Cam4 belonged to P. t. vellerosus. Genetic analyses of the viruses together with serological data demonstrated that at least one of the two P. t. troglodytes chimpanzees (Cam5) was infected in the wild, and revealed a horizontal transmission between Cam3 and Cam4. These data confirm that P. t. troglodytes is a natural host for HIV-1-related viruses. Furthermore, they show that SIVcpz can be transmitted in captivity, from one chimpanzee subspecies to another. All three SIVcpz-cam viruses clustered with HIV-1 N in env. The full Cam3 SIVcpz genome sequence showed a very close phylogenetic relationship with SIVcpz-US, a virus identified in a P. t. troglodytes chimpanzee captured nearly 40 years earlier. Like SIVcpz-US, SIVcpz-cam3 was closely related to HIV-1 N in env, but not in pol, supporting the hypothesis that HIV-1 N results from a recombination event. SIVcpz from chimpanzees born in the wild in Cameroon are thus strongly related in env to HIV-1 N from Cameroon, demonstrating the geographic coincidence of these human and simian viruses and providing a further strong argument in favor of the origin of HIV-1 being in chimpanzees.     

Mono- and Dialkyl Glycerol Ether Lipids in Anaerobic Bacteria: Biosynthetic Insights from the Mesophilic Sulfate Reducer Desulfatibacillum alkenivorans PF2803T

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803^T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1′-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803^T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1′-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.     

Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo-Inositol Accumulation

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants.     

Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of: 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating: a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.     

MGOUN3, an Arabidopsis gene with TetratricoPeptide-Repeat-related motifs, regulates meristem cellular organization

In order to understand the functioning of apical meristems in Arabidopsis more clearly, a new mutant, mgoun3 (mgo3), affected in the structural organization and the functional regulation of both shoot and root meristems has been isolated. mgo3 plants display perturbations in leaf morphogenesis, in the spatial and the temporal formation of primordia, and frequent fasciation of the inflorescence stem. Cellular analysis showed that both cellular organization and cell identity patterning are impaired in the mutant meristems. The MGO3 gene has been isolated by positional cloning. The protein deduced from the cDNA sequence contains TetratricoPeptide Repeats (TPR) and Leucine-Rich Repeats (LRR), two motifs that are thought to act in protein-protein interactions. This gene appears to be unique in the Arabidopsis genome. Although the MGO3 protein presents TPR as in the Arabidopsis proteins HOBBIT and SPINDLY, the MGO3 motifs are more similar to those present in LGN-related proteins, which are regulators for some of the asymmetric cell divisions in animal development. These features suggest a key role for MGO3 in meristematic cell divisions and would be of interest for the comparison between plant and animal development.     

MGOUN3: evidence for chromatin-mediated regulation of FLC expression

The MGOUN3(MGO3)/BRUSHY1(BRU1)/TONSOKU(TSK) gene of Arabidopsis thaliana encodes a nuclear leucine-glycine-asparagine (LGN) domain protein that may be implicated in chromatin dynamics and genome maintenance. Mutants with defects in MGO3 display a fasciated stem and disorganized meristem structures. The transition to flowering was examined in mgo3 mutants and it was found that, under short days, the mutants flowered significantly earlier than the wild-type plants. Study of flowering-time associated gene expression showed that the floral transition inhibitor gene FLC was under-expressed in the mutant background. Ectopic expression of the flower-specific genes AGAMOUS (AG), PISTILLATA (PI), and SEPALLATA3 (SEP3) in mgo3 vegetative organs was also detected. Western blot and chromatin immunoprecipitation experiments suggested that histone H3 acetylation may be altered in the mgo3 background. Together, these data suggest that MGO3 is required for the correct transition to flowering and that this may be mediated by histone acetylation and associated changes in FLC expression.