Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release.

This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.

Arginine reprograms metabolism in liver cancer via RBM39.

Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in murine and patient hepatocellular carcinoma (HCC), despite reduced expression of arginine synthesis genes. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Plasticity of the adult human small intestinal stoma microbiota.

The human distal small intestine (ileum) has a distinct microbiota, but human studies investigating its composition and function have been limited by the inaccessibility of the ileum without purging and/or deep intubation. We investigated inherent instability, temporal dynamics, and the contribution of fed and fasted states using stoma samples from cured colorectal cancer patients as a non-invasive access route to the otherwise inaccessible small and large intestines. Sequential sampling of the ileum before and after stoma formation indicated that ileostoma microbiotas represented that of the intact small intestine. Ileal and colonic stoma microbiotas were confirmed as distinct, and two types of instability in ileal host-microbial relationships were observed: inter-digestive purging followed by the rapid postprandial blooming of bacterial biomass and sub-strain appearance and disappearance within individual taxa after feeding. In contrast to the relative stability of colonic microbiota, the human small intestinal microbiota biomass and its sub-strain composition can be highly dynamic.

Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly.

Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.

Cooperation and Cheating among Germinating Spores.

Many microbes produce stress-resistant spores to survive unfavorable conditions [1-4] and enhance dispersal [1, 5]. Cooperative behavior is integral to the process of spore formation in some species [3, 6], but the degree to which germination of spore populations involves social interactions remains little explored. Myxococcus xanthus is a predatory soil bacterium that upon starvation forms spore-filled multicellular fruiting bodies that often harbor substantial diversity of endemic origin [7, 8]. Here we demonstrate that germination of M. xanthus spores formed during fruiting-body development is a social process involving at least two functionally distinct social molecules. Using pairs of natural isolates each derived from a single fruiting body that emerged on soil, we first show that spore germination exhibits positive density dependence due to a secreted "public-good" germination factor. Further, we find that a germination defect of one strain under saline stress in pure culture is complemented by addition of another strain that germinates well in saline environments and mediates cheating by the defective strain. Glycine betaine, an osmo-protectant utilized in all domains of life, is found to mediate saline-specific density dependence and cheating. Density dependence in non-saline conditions is mediated by a distinct factor, revealing socially complex spore germination involving multiple social molecules.

Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation.

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism-two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

ß-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant-although lower-level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells' response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells' immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via ß-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and ß-oxidation as important energy providers during acute glucose starvation.

Systematic mapping of protein-metabolite interactions in central metabolism of Escherichia coli.

Metabolite binding to proteins regulates nearly all cellular processes, but our knowledge of these interactions originates primarily from empirical in vitro studies. Here, we report the first systematic study of interactions between water-soluble proteins and polar metabolites in an entire biological subnetwork. To test the depth of our current knowledge, we chose to investigate the well-characterized Escherichia coli central metabolism. Using ligand-detected NMR, we assayed 29 enzymes towards binding events with 55 intracellular metabolites. Focusing on high-confidence interactions at a false-positive rate of 5%, we detected 98 interactions, among which purine nucleotides accounted for one-third, while 50% of all metabolites did not interact with any enzyme. In contrast, only five enzymes did not exhibit any metabolite binding and some interacted with up to 11 metabolites. About 40% of the interacting metabolites were predicted to be allosteric effectors based on low chemical similarity to their target's reactants. For five of the eight tested interactions, in vitro assays confirmed novel regulatory functions, including ATP and GTP inhibition of the first pentose phosphate pathway enzyme. With 76 new candidate regulatory interactions that have not been reported previously, we essentially doubled the number of known interactions, indicating that the presently available information about protein-metabolite interactions may only be the tip of the iceberg.

The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of σ54 -RNA polymerase.

DmpR is the obligate transcriptional activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR belongs to the AAA+ class of mechano-transcriptional regulators that employ ATP-hydrolysis to engage and remodel σ54 -RNA polymerase to allow transcriptional initiation. Previous work has established that binding of phenolic effectors by DmpR is a prerequisite to relieve interdomain repression and allow ATP-binding to trigger transition to its active multimeric conformation, and further that a structured interdomain linker between the effector- and ATP-binding domains is involved in coupling these processes. Here, we present evidence from ATPase and in vivo and in vitro transcription assays that a tyrosine residue of the interdomain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and aromatic effector-responsive transcriptional activation by DmpR. An alanine substitution of Y233A results in both increased ATPase activity and enhanced sensitivity to aromatic effectors. We propose a model in which effector-binding relocates Y233 to synchronize signal-reception with multimerisation to provide physiologically appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family and has implications for development of enhanced sensitivity of biosensor used to detect pollutants.

Molecular mechanism of ATP versus GTP selectivity of adenylate kinase.

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.

Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7210.

Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (CP) and, if present, the resolving Cys (CR). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 Å resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.

Structural basis for ligand binding to an enzyme by a conformational selection pathway.

Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-Å X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.

PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis.

Sensing and response to changes in nutrient availability are essential for the lifestyle of environmental and pathogenic bacteria. Serine/threonine protein kinase G (PknG) is required for virulence of the human pathogen Mycobacterium tuberculosis, and its putative substrate GarA regulates the tricarboxylic acid cycle in M. tuberculosis and other Actinobacteria by protein-protein binding. We sought to understand the stimuli that lead to phosphorylation of GarA, and the roles of this regulatory system in pathogenic and non-pathogenic bacteria. We discovered that M. tuberculosis lacking garA was severely attenuated in mice and macrophages and furthermore that GarA lacking phosphorylation sites failed to restore the growth of garA deficient M. tuberculosis in macrophages. Additionally we examined the impact of genetic disruption of pknG or garA upon protein phosphorylation, nutrient utilization and the intracellular metabolome. We found that phosphorylation of GarA requires PknG and depends on nutrient availability, with glutamate and aspartate being the main stimuli. Disruption of pknG or garA caused opposing effects on metabolism: a defect in glutamate catabolism or depletion of intracellular glutamate, respectively. Strikingly, disruption of the phosphorylation sites of GarA was sufficient to recapitulate defects caused by pknG deletion. The results suggest that GarA is a cellular target of PknG and the metabolomics data demonstrate that the function of this signaling system is in metabolic regulation. This function in amino acid homeostasis is conserved amongst the Actinobacteria and provides an example of the close relationship between metabolism and virulence.

Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria.

Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.

Systematic Identification of Protein-Metabolite Interactions in Complex Metabolite Mixtures by Ligand-Detected Nuclear Magnetic Resonance Spectroscopy.

Protein-metabolite interactions play a vital role in the regulation of numerous cellular processes. Consequently, identifying such interactions is a key prerequisite for understanding cellular regulation. However, the noncovalent nature of the binding between proteins and metabolites has so far hampered the development of methods for systematically mapping protein-metabolite interactions. The few available, largely mass spectrometry-based, approaches are restricted to specific metabolite classes, such as lipids. In this study, we address this issue and show the potential of ligand-detected nuclear magnetic resonance (NMR) spectroscopy, which is routinely used in drug development, to systematically identify protein-metabolite interactions. As a proof of concept, we selected four well-characterized bacterial and mammalian proteins (AroG, Eno, PfkA, and bovine serum albumin) and identified metabolite binders in complex mixes of up to 33 metabolites. Ligand-detected NMR captured all of the reported protein-metabolite interactions, spanning a full range of physiologically relevant Kd values (low micromolar to low millimolar). We also detected a number of novel interactions, such as promiscuous binding of the negatively charged metabolites citrate, AMP, and ATP, as well as binding of aromatic amino acids to AroG protein. Using in vitro enzyme activity assays, we assessed the functional relevance of these novel interactions in the case of AroG and show that l-tryptophan, l-tyrosine, and l-histidine act as novel inhibitors of AroG activity. Thus, we conclude that ligand-detected NMR is suitable for the systematic identification of functionally relevant protein-metabolite interactions.

Treosulfan in the Treatment of Advanced Ovarian Cancer - Results of a German Multicenter Non-interventional Study.

BACKGROUND: Data on routine systemic treatment of patients with ovarian cancer are currently available only to a limited degree. The alkylating agent treosulfan is approved in oral (p.o.) and intravenous (i.v.) form for the treatment of ovarian carcinoma. The present non-interventional study analyzed the clinical use of treosulfan in Germany, evaluating the mode of application, toxicity, and response and survival rate. PATIENTS AND METHODS: Two hundred and forty-eight ovarian cancer patients in 57 Centers, who received treosulfan mainly either i.v. (5,000-8,000 mg/m(2) d1, q21d or q28d) or p.o. (400-600 mg/m(2) d1-14 or 21, q28d) for at least one therapy cycle were evaluable and were included in the study. RESULTS: With a median age of 70 years (range=36-92 years), predominantly elderly patients received treosulfan treatment. Most participants presented serous histology (131, 52.8%) and advanced-stage FIGO III (122, 49%) or IV (55, 22%) disease. Median ECOG status was 1 (range=0-2), whereas cardiac co-morbidity was common (31%). Treosulfan was usually administered as second- (26%), third- (21%) or fourth-line (17%) therapy. Two hundred and one patients received i.v. and 47 p.o. TREATMENT: The most common reason for dose modifications was due to hematological toxicity (46%). The main reason for a therapy discontinuation was progressive disease (38.5%). Response was observed in 25.8% of participants, disease stabilization in 28.6 % and progress in 45.6%. The median progression-free and overall survival was 196 and 405 days, respectively. CONCLUSION: In predominantly elderly and heavily pre-treated patients with recurrent ovarian cancer, treosulfan featured a clinical relevant efficacy and well-manageable, mostly hematological, toxicity, which resulted in a positive therapeutic index.

Real-time metabolome profiling of the metabolic switch between starvation and growth.

Metabolic systems are often the first networks to respond to environmental changes, and the ability to monitor metabolite dynamics is key for understanding these cellular responses. Because monitoring metabolome changes is experimentally tedious and demanding, dynamic data on time scales from seconds to hours are scarce. Here we describe real-time metabolome profiling by direct injection of living bacteria, yeast or mammalian cells into a high-resolution mass spectrometer, which enables automated monitoring of about 300 compounds in 15-30-s cycles over several hours. We observed accumulation of energetically costly biomass metabolites in Escherichia coli in carbon starvation-induced stationary phase, as well as the rapid use of these metabolites upon growth resumption. By combining real-time metabolome profiling with modeling and inhibitor experiments, we obtained evidence for switch-like feedback inhibition in amino acid biosynthesis and for control of substrate availability through the preferential use of the metabolically cheaper one-step salvaging pathway over costly ten-step de novo purine biosynthesis during growth resumption.

Structural basis for catalytically restrictive dynamics of a high-energy enzyme state.

An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or 'invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21.

The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.