Molecular analysis of the prokaryotic ubiquitin‐like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.     

The Mechanism of Mycobacterium smegmatis PafA Self-Pupylation

PafA, the prokaryotic ubiquitin-like protein (Pup) ligase, catalyzes the Pup modification of bacterial proteins and targets the substrates for proteasomal degradation. It has been reported that that M. smegmatis PafA can be poly-pupylated. In this study, the mechanism of PafA self-pupylation is explored. We found that K320 is the major target residue for the pupylation of PafA. During the self-pupylation of PafA, the attachment of the first Pup to PafA is catalyzed by the other PafA molecule through an intermolecular reaction, while the formation of the polymeric Pup chain is carried out in an intramolecular manner through the internal ligase activity of the already pupylated PafA. Among the three lysine residues, K7, K31 and K61, in M. smegmatis Pup, K7 and K31 are involved in the formation of the poly-Pup chain in PafA poly-pupylation. Poly-pupylation of PafA can be reversibly regulated by depupylase Dop. The polymeric Pup chain formed through K7/K31 linkage is much more sensitive to Dop than the mono-Pup directly attached to PafA. Moreover, self-pupylation of PafA is involved in the regulation of its stability in vivo in a proteasome-dependent manner, suggesting that PafA self-pupylation functions as a mechanism in the auto-regulation of the Pup-proteasome system.     

Identification of substrates of the Mycobacterium tuberculosis proteasome

The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.     

Activity of the mycobacterial proteasomal ATPase Mpa is reversibly regulated by pupylation

Pupylation is a bacterial post-translational modification of target proteins on lysine residues with prokaryotic ubiquitin-like protein Pup. Pup-tagged substrates are recognized by a proteasome-interacting ATPase termed Mpa in Mycobacterium tuberculosis. Mpa unfolds pupylated substrates and threads them into the proteasome core particle for degradation. Interestingly, Mpa itself is also a pupylation target. Here, we show that the Pup ligase PafA predominantly produces monopupylated Mpa modified homogeneously on a single lysine residue within its C-terminal region. We demonstrate that this modification renders Mpa functionally inactive. Pupylated Mpa can no longer support Pup-mediated proteasomal degradation due to its inability to associate with the proteasome core. Mpa is further inactivated by rapid Pup- and ATPase-driven deoligomerization of the hexameric Mpa ring. We show that pupylation of Mpa is chemically and functionally reversible. Mpa regains its enzymatic activity upon depupylation by the depupylase Dop, affording a rapid and reversible activity control over Mpa function.     

Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo

Proteasome‐bearing bacteria make use of a ubiquitin‐like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C‐terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady‐state levels of two known proteasomal substrates are drastically increased. Pupylation can be re‐established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C‐terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N‐terminal ATP‐binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.     

Distinct Conformations of a Putative Translocation Element in Poliovirus Polymerase

The mechanism whereby RNA is translocated by the single subunit viral RNA-dependent RNA polymerases is not yet understood. These enzymes lack homologs of the “O-helix” structures and associated fingers domain movements thought to be responsible for translocation in many DNA-templated polymerases. The structures of multiple picornavirus polymerase elongation complexes suggest that these enzymes use a different molecular mechanism where translocation is not strongly coupled to the opening of the active site following catalysis. Here we present the 2.0- to 2.6-Å-resolution crystal structures and biochemical data for 12 poliovirus polymerase mutants that together show how proper enzyme functions and translocation activity requires conformational flexibility of a loop sequence in the palm domain B-motif. Within the loop, the Ser288-Gly289-Cys290 sequence is shown to play a major role in the catalytic cycle based on RNA binding, processive elongation activity, and single nucleotide incorporation assays. The structures show that Ser288 forms a key hydrogen bond with Asp238, the backbone flexibility of Gly289 is required for translocation competency, and Cys290 modulates the overall elongation activity of the enzyme. Some conformations of the loop represent likely intermediates on the way to forming the catalytically competent closed active site, while others are consistent with a role in promoting translocation of the nascent base pair out of the active site. The loop structure and key residues surrounding it are highly conserved, suggesting that the structural dynamics we observe in poliovirus 3D^pol are a common feature of viral RNA-dependent RNA polymerases.     

Conserved residues of liquefying alpha-amylases are concentrated in the vicinity of active site

Three dimensional structure of three liquefying type Bacillus alpha-amylases were modeled based on sequence analyses and refined structure of Aspergillus oryzae enzyme. The models suggest that the overall folding motif of alpha-amylases is conserved. The active site, substrate binding and stabilizing calcium binding residues are conserved and concentrated in a cleft between two domains. They constitute the core of alpha-amylases to which other, less conserved regions are attached. The bacterial enzymes have a loop of about 45 residues near the active site and Ca2+ binding region. The loop may be important for the liquefying function of these enzymes.     

Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of d-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.     

Crystal structures of a poxviral glutaredoxin in the oxidized and reduced states show redox-correlated structural changes

Glutaredoxins act as reducing agents for the large subunit of ribonucleotide reductase (R1) in many prokaryotes and eukaryotes, including humans. The same relationship has been proposed for the glutaredoxin and R1 proteins expressed by all orthopoxviruses, including vaccinia, variola, and ectromelia virus. Interestingly, the orthopoxviral proteins share 45% and 78% sequence identity with human glutaredoxin-1 (Grx-1) and R1, respectively. To study structure-function relationships of the vertebrate Grx-1 family, and reveal potential viral adaptations, we have determined crystal structures of the ectromelia virus glutaredoxin, EVM053, in the oxidized and reduced states. The structures show a large redox-induced conformational rearrangement of Tyr21 and Thr22 near the active site. We predict that the movement of Tyr21 is a viral-specific adaptation that increases the redox potential by stabilizing the reduced state. The conformational switch of Thr22 appears to be shared by vertebrate Grx-1 and may affect the strictly conserved Lys20. A crystal packing-induced structural change in residues 68-70 affects the GSH-binding loop, and our structures reveal a potential interaction network that connects the GSH-binding loop and the active site. EVM053 also exhibits a novel cis-proline (Pro53) in a loop that has been shown to contribute to R1-binding in Escherichia coli Grx-1. The cis-peptide bond of Pro53 may be required to promote electrostatic interactions between Lys52 and the C-terminal carboxylate of R1. Finally, dimethylarsenite was covalently attached to Cys23 in one reduced EVM053 structure and our preliminary data show that EVM053 has dimethylarsenate reductase activity.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Dop functions as a depupylase in the prokaryotic ubiquitin‐like modification pathway

Post‐translational modification of proteins with prokaryotic ubiquitin‐like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two‐step process in which the carboxy‐terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ‐carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.     

Sequences required for the activity of PTOX (IMMUTANS), a plastid terminal oxidase: in vitro and in planta mutagenesis of iron-binding sites and a conserved sequence that corresponds to Exon 8

The thylakoid membranes of most photosynthetic organisms contain a terminal oxidase (PTOX, the product of the Arabidopsis IMMUTANS gene) that functions in the oxidation of the plastoquinone pool. PTOX and AOX are diiron carboxylate proteins, and based on crystal structures of other members of this protein class, a structural model of PTOX has been proposed in which the ligation sphere of the diiron center is composed of six conserved histidine and glutamate residues. We tested the functional significance of these residues by site-directed mutagenesis of PTOX in vitro and in planta, taking advantage null immutans alleles for the latter studies. These experiments showed that the six iron-binding sites do not tolerate change, even conservative ones. We also examined the significance of a conserved sequence in (or near) the PTOX active site that corresponds precisely to Exon 8 of the IM gene. In vitro and in planta mutagenesis revealed that conserved amino acids within this domain can be altered but that deletion of all or part of the domain abolishes activity. Because protein accumulates normally in the deletion mutants, the data suggest that the conformation of the Exon 8 sequence is important for PTOX activity. An allele of immutans (designated 3639) was identified that lacks the Exon 8 sequence; it does not accumulate PTOX protein. Chloroplast import assays revealed that mutant enzymes lacking Exon 8 have enhanced turnover. We conclude that the Exon 8 domain is required not only for PTOX activity but also for its stability.     

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.     

Triggering loops and enzyme function: identification of loops that trigger and modulate movements

Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.     

Allosteric loss-of-function mutations in HIV-1 Nef from a long-term non-progressor

Activation of Src family kinases by human immunodeficiency virus type 1 (HIV-1) Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site influence Nef interactions allosterically with a key effector protein linked to AIDS progression.     

Identification and Characterization of γ-Glutamylamine Cyclotransferase,an Enzyme Responsible for γ-Glutamyl-?-lysine Catabolism

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.     

Conformational analysis and clustering of short and medium size loops connecting regular secondary structures: a database for modeling and prediction.

Loops are regions of nonrepetitive conformation connecting regular secondary structures. We identified 2,024 loops of one to eight residues in length, with acceptable main-chain bond lengths and peptide bond angles, from a database of 223 protein and protein-domain structures. Each loop is characterized by its sequence, main-chain conformation, and relative disposition of its bounding secondary structures as described by the separation between the tips of their axes and the angle between them. Loops, grouped according to their length and type of their bounding secondary structures, were superposed and clustered into 161 conformational classes, corresponding to 63% of all loops. Of these, 109 (51% of the loops) were populated by at least four nonhomologous loops or four loops sharing a low sequence identity. Another 52 classes, including 12% of the loops, were populated by at least three loops of low sequence similarity from three or fewer nonhomologous groups. Loop class suprafamilies resulting from variations in the termini of secondary structures are discussed in this article. Most previously described loop conformations were found among the classes. New classes included a 2:4 type IV hairpin, a helix-capping loop, and a loop that mediates dinucleotide-binding. The relative disposition of bounding secondary structures varies among loop classes, with some classes such as beta-hairpins being very restrictive. For each class, sequence preferences as key residues were identified; those most frequently at these conserved positions than in proteins were Gly, Asp, Pro, Phe, and Cys. Most of these residues are involved in stabilizing loop conformation, often through a positive phi conformation or secondary structure capping. Identification of helix-capping residues and beta-breakers among the highly conserved positions supported our decision to group loops according to their bounding secondary structures. Several of the identified loop classes were associated with specific functions, and all of the member loops had the same function; key residues were conserved for this purpose, as is the case for the parvalbumin-like calcium-binding loops. A significant number, but not all, of the member loops of other loop classes had the same function, as is the case for the helix-turn-helix DNA-binding loops. This article provides a systematic and coherent conformational classification of loops, covering a broad range of lengths and all four combinations of bounding secondary structure types, and supplies a useful basis for modelling of loop conformations where the bounding secondary structures are known or reliably predicted.     

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).     

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.