Legionella para-effectors target chromatin and promote bacterial replication.

Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a ∆lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout (∆lphD∆romA) can restore intracellular replication. Uniquely, we present evidence for "para-effectors", an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens has the potential to lead to the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences.

Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: Molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host.

New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach.

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.

Structure of the RZZ complex and molecular basis of its interaction with Spindly.

Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αß'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod-Zwilch-ZW10 (RZZ) complex.

The spindle-assembly checkpoint (SAC) monitors kinetochore-microtubule attachment during mitosis. In metazoans, the three-subunit Rod-Zwilch-ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1-Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P31 (No. 144) or P32 (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = ß = 90.0, γ = 120.0°.

The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation.

Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases.

Towards elucidating the tubulin code.

Genetically encoded and post-translationally generated variations of tubulin C-terminal tails give rise to extensive heterogeneity of the microtubule cytoskeleton. The generation of different tubulin variants in yeast now demonstrates how single amino-acid differences or post-translational modifications can modulate the behaviour of selected molecular motors.

Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction.

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR-KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

Functional plasticity and allosteric regulation of α-ketoglutarate decarboxylase in central mycobacterial metabolism.

The α-ketoglutarate dehydrogenase (KDH) complex is a major regulatory point of aerobic energy metabolism. Mycobacterium tuberculosis was reported to lack KDH activity, and the putative KDH E1o component, α-ketoglutarate decarboxylase (KGD), was instead assigned as a decarboxylase or carboligase. Here, we show that this protein does in fact sustain KDH activity, as well as the additional two reactions, and these multifunctional properties are shared by the Escherichia coli homolog, SucA. We also show that the mycobacterial enzyme is finely regulated by an additional acyltransferase-like domain and by the action of acetyl-CoA, a powerful allosteric activator able to enhance the concerted protein motions observed during catalysis. Our results uncover the functional plasticity of a crucial node in bacterial metabolism, which may be important for M. tuberculosis during host infection.

Structural analysis of the RZZ complex reveals common ancestry with multisubunit vesicle tethering machinery.

The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal beta propeller followed by an alpha solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores.

The FHA-containing protein GarA acts as a phosphorylation-dependent molecular switch in mycobacterial signaling.

Fork-head associated (FHA) domains are widely found in bacteria, but their cellular functions remain unclear. Here, we focus on Mycobacterium tuberculosis GarA, an FHA-containing protein conserved in actinomycetes that is phosphorylated by different Ser/Thr protein kinases. Using various physicochemical approaches, we show that phosphorylation significantly stabilizes GarA, and that its FHA domain interacts strongly with the phosphorylated N-terminal extension. Altogether, our results indicate that phosphorylation triggers an intra-molecular protein closure, blocking the phosphothreonine-binding site and switching off the regulatory properties of GarA. The model can explain the reported functions of this mycobacterial protein as regulator of glycogen degradation and glutamate metabolism.

Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential.

Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses 'eukaryotic-like' Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds.

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases.

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the alpha isoform of human PP2C (PP2Calpha), which folds into an alpha/beta domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn(2+)-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.

Insights into the catalytic mechanism of PPM Ser/Thr phosphatases from the atomic resolution structures of a mycobacterial enzyme.

Serine/threonine-specific phosphatases (PPs) represent, after protein tyrosine phosphatases, the second major class of enzymes that catalyze the dephosphorylation of proteins. They are classed in two large families, known as PPP and PPM, on the basis of sequence similarities, metal ion dependence, and inhibitor sensitivity. Despite their wide species distribution and broad physiological roles, the catalytic mechanism of PPM phosphatases has been primarily inferred from studies of a single enzyme, human PP2Calpha. Here, we report the biochemical characterization and the atomic resolution structures of a soluble PPM phosphatase from the saprophyte Mycobacterium smegmatis in complex with different ligands. The structures provide putative snapshots along the catalytic cycle, which support an associative reaction mechanism that differs in some important aspects from the currently accepted model and reinforces the hypothesis of convergent evolution in PPs.

The crystal structure of M. leprae ML2640c defines a large family of putative S-adenosylmethionine-dependent methyltransferases in mycobacteria.

Mycobacterium leprae protein ML2640c belongs to a large family of conserved hypothetical proteins predominantly found in mycobacteria, some of them predicted as putative S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTase). As part of a Structural Genomics initiative on conserved hypothetical proteins in pathogenic mycobacteria, we have determined the structure of ML2640c in two distinct crystal forms. As expected, ML2640c has a typical MTase core domain and binds the methyl donor substrate AdoMet in a manner consistent with other known members of this structural family. The putative acceptor substrate-binding site of ML2640c is a large internal cavity, mostly lined by aromatic and aliphatic side-chain residues, suggesting that a lipid-like molecule might be targeted for catalysis. A flap segment (residues 222-256), which isolates the binding site from the bulk solvent and is highly mobile in the crystal structures, could serve as a gateway to allow substrate entry and product release. The multiple sequence alignment of ML2640c-like proteins revealed that the central alpha/beta core and the AdoMet-binding site are very well conserved within the family. However, the amino acid positions defining the binding site for the acceptor substrate display a higher variability, suggestive of distinct acceptor substrate specificities. The ML2640c crystal structures offer the first structural glimpses at this important family of mycobacterial proteins and lend strong support to their functional assignment as AdoMet-dependent methyltransferases.

The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria.

Mycobacterium tuberculosis PknB is an essential receptor-like protein kinase involved in cell growth control. Here, we demonstrate that mitoxantrone, an anthraquinone derivative used in cancer therapy, is a PknB inhibitor capable of preventing mycobacterial growth. The structure of the complex reveals that mitoxantrone partially occupies the adenine-binding pocket in PknB, providing a framework for the design of compounds with potential therapeutic applications. PknB crystallizes as a 'back-to-back' homodimer identical to those observed in other structures of PknB in complex with ATP analogs. This organization resembles that of the RNA-dependent protein kinase PKR, suggesting a mechanism for kinase activation in mycobacteria.

Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases.

The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four 'receptor-like' protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26-100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.

Altered substrate specificity in flavocytochrome b2: structural insights into the mechanism of L-lactate dehydrogenation.

Flavocytochrome b(2) from Saccharomyces cerevisiae is a l-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301, 829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate l-mandelate than it is with l-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 A resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 A resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

Sialidases are a superfamily of sialic-acid-releasing enzymes that are of significant interest due to their implication as virulence factors in the pathogenesis of a number of diseases. However, extensive studies of viral and microbial sialidases have failed to provide a comprehensive picture of their mechanistic properties, in part because the structures of competent enzyme-substrate complexes and reaction intermediates have never been described. Here we report these structures for the Trypanosoma cruzi trans-sialidase (TcTS), showing that catalysis by sialidases occurs via a similar mechanism to that of other retaining glycosidases, but with some intriguing differences that may have evolved in response to the substrate structure.