The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities

Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite's cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90 alpha or Hsp90 beta, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90's. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis

The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.

Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis{triangledown}

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3',5'-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the {Delta}MSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the {Delta}MSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the {Delta}MSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M. tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the selfbiotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over selfbiotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-59 AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

Functional specificity among yeast mitochondrial Hsp70s paralogs and orthologs

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can “make or break” mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance

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.

Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THE SHMT also catalyses several THF-independent side reactions such as cleavage of P-hydroxy amino acids, trans-amination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect oil either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a Mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three Mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

Background The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms. Results The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies. Conclusion The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials.

Identification of putative regulatory motifs in the upstream regions of co-expressed functional groups of genes in Plasmodium falciparum

Background: Regulation of gene expression in Plasmodium falciparum (Pf) remains poorly understood. While over half the genes are estimated to be regulated at the transcriptional level, few regulatory motifs and transcription regulators have been found. Results: The study seeks to identify putative regulatory motifs in the upstream regions of 13 functional groups of genes expressed in the intraerythrocytic developmental cycle of Pf. Three motif-discovery programs were used for the purpose, and motifs were searched for only on the gene coding strand. Four motifs – the 'G-rich', the 'C-rich', the 'TGTG' and the 'CACA' motifs – were identified, and zero to all four of these occur in the 13 sets of upstream regions. The 'CACA motif' was absent in functional groups expressed during the ring to early trophozoite transition. For functional groups expressed in each transition, the motifs tended to be similar. Upstream motifs in some functional groups showed 'positional conservation' by occurring at similar positions relative to the translational start site (TLS); this increases their significance as regulatory motifs. In the ribonucleotide synthesis, mitochondrial, proteasome and organellar translation machinery genes, G-rich, C-rich, CACA and TGTG motifs, respectively, occur with striking positional conservation. In the organellar translation machinery group, G-rich motifs occur close to the TLS. The same motifs were sometimes identified for multiple functional groups; differences in location and abundance of the motifs appear to ensure different modes of action. Conclusion: The identification of positionally conserved over-represented upstream motifs throws light on putative regulatory elements for transcription in Pf.

Some cytoplasmic details of yeast revealed by the electron microscope

Abstract is not available.