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1.
Nat Commun ; 15(1): 6076, 2024 Jul 19.
Article in English | MEDLINE | ID: mdl-39025835

ABSTRACT

Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Drug Synergism , Lung Neoplasms , Mechanistic Target of Rapamycin Complex 1 , Proto-Oncogene Proteins p21(ras) , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Animals , Cell Line, Tumor , Mice , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Xenograft Model Antitumor Assays , Signal Transduction/drug effects , Cyclin D1/metabolism , Cyclin D1/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Female , Bcl-2-Like Protein 11/metabolism , Bcl-2-Like Protein 11/genetics
3.
Nat Commun ; 13(1): 6185, 2022 10 19.
Article in English | MEDLINE | ID: mdl-36261450

ABSTRACT

Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both reactions, an initial biotin carboxylation and the subsequent carboxyl transfer to pyruvate substrate to produce oxaloacetate. Reaction sites are at long distance, and there are several co-factors that play as allosteric regulators. Here, using cryoEM we explore the structure of active PC tetramers focusing on active sites and on the conformational space of the oligomers. The results capture the mobile domain at both active sites and expose catalytic steps of both reactions at high resolution, allowing the identification of substrates and products. The analysis of catalytically active PC tetramers reveals the role of certain motions during enzyme functioning, and the structural changes in the presence of additional cofactors expose the mechanism for allosteric regulation.


Subject(s)
Biotin , Pyruvate Carboxylase , Pyruvate Carboxylase/genetics , Cryoelectron Microscopy , Oxaloacetates , Pyruvic Acid
4.
J Bacteriol ; 203(19): e0008021, 2021 09 08.
Article in English | MEDLINE | ID: mdl-34309402

ABSTRACT

3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. IMPORTANCE c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.


Subject(s)
Biosensing Techniques , Dinucleoside Phosphates/chemistry , Fluorescence Resonance Energy Transfer , Nucleotides/metabolism , Bacillus subtilis/chemistry , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dinucleoside Phosphates/metabolism , Gene Expression Regulation, Bacterial/physiology , Listeria monocytogenes/metabolism , Models, Molecular , Mutation , Protein Conformation
5.
Sci Rep ; 11(1): 277, 2021 01 11.
Article in English | MEDLINE | ID: mdl-33431973

ABSTRACT

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.


Subject(s)
Biocatalysis , Carbon-Nitrogen Lyases/chemistry , Carbon-Nitrogen Lyases/metabolism , Eurotiales/enzymology , Biodegradation, Environmental , Catalytic Domain , Crystallography, X-Ray , Kinetics , Models, Molecular , Temperature
6.
Proc Natl Acad Sci U S A ; 114(35): E7226-E7235, 2017 08 29.
Article in English | MEDLINE | ID: mdl-28808024

ABSTRACT

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.


Subject(s)
Dinucleoside Phosphates/metabolism , Pyruvate Carboxylase/metabolism , Pyruvate Carboxylase/physiology , Adenosine Monophosphate/metabolism , Aspartic Acid/biosynthesis , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray/methods , Cyclic AMP/metabolism , Dinucleoside Phosphates/physiology , Lactobacillales/metabolism , Lactococcus lactis/metabolism , Protein Conformation , Second Messenger Systems/physiology , Structure-Activity Relationship
7.
Mol Microbiol ; 104(2): 212-233, 2017 04.
Article in English | MEDLINE | ID: mdl-28097715

ABSTRACT

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the ß-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.


Subject(s)
Dinucleoside Phosphates/metabolism , Listeria monocytogenes/metabolism , Acetyl Coenzyme A/metabolism , Bacterial Proteins/metabolism , Cell Wall/metabolism , Dinucleoside Phosphates/genetics , Dinucleoside Phosphates/physiology , Drug Resistance, Microbial , Gene Expression Regulation, Bacterial/genetics , Listeria monocytogenes/growth & development , Osmoregulation/physiology , Osmotic Pressure , Phosphorus-Oxygen Lyases/metabolism , Pyruvate Carboxylase/metabolism , Second Messenger Systems , Suppression, Genetic
8.
Nat Commun ; 7: 12713, 2016 10 06.
Article in English | MEDLINE | ID: mdl-27708276

ABSTRACT

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the ß subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4ß4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2ß4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis.


Subject(s)
Bacterial Proteins/chemistry , Methylobacillus/enzymology , Pyruvate Carboxylase/chemistry , Acetyl-CoA Carboxylase/chemistry , Biotin/chemistry , Carbon-Nitrogen Ligases/chemistry , Crystallography, X-Ray , Escherichia coli/metabolism , Fatty Acid Synthase, Type II/chemistry , Gene Deletion , Holoenzymes , Mutagenesis , Mutagenesis, Site-Directed , Mutation , Phenotype , Protein Conformation , Protein Domains , Pseudomonas aeruginosa/enzymology
9.
Mol Microbiol ; 102(2): 233-243, 2016 10.
Article in English | MEDLINE | ID: mdl-27378384

ABSTRACT

Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c-di-AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c-di-AMP content and that c-di-AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c-di-AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/metabolism , Dinucleoside Phosphates/metabolism , Listeria monocytogenes/cytology , Listeria monocytogenes/metabolism , Adenosine Monophosphate/metabolism , Betaine/metabolism , Biological Transport, Active , Carnitine/metabolism , Cyclic AMP/metabolism , Cystathionine beta-Synthase/metabolism , Osmolar Concentration , Osmotic Pressure/physiology
10.
Microbiologyopen ; 4(3): 361-74, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25693966

ABSTRACT

Cyclic-di-AMP (c-di-AMP) is a broadly conserved bacterial second messenger that is of importance in bacterial physiology. The molecular receptors mediating the cellular responses to the c-di-AMP signal are just beginning to be discovered. PstA is a previously uncharacterized PII -like protein which has been identified as a c-di-AMP receptor. PstA is widely distributed and conserved among Gram-positive bacteria in the phylum Firmicutes. Here, we report the biochemical, structural, and functional characterization of PstA from Listeria monocytogenes. We have determined the crystal structures of PstA in the c-di-AMP-bound and apo forms at 1.6 and 2.9 Å resolution, respectively, which provide the molecular basis for its specific recognition of c-di-AMP. PstA forms a homotrimer structure that has overall similarity to the PII protein family which binds ATP. However, PstA is markedly different from PII proteins in the loop regions, and these structural differences mediate the specific recognition of their respective nucleotide ligand. The residues composing the c-di-AMP binding pocket are conserved, suggesting that c-di-AMP recognition by PstA is of functional importance. Disruption of pstA in L. monocytogenes affected c-di-AMP-mediated alterations in bacterial growth and lysis. Overall, we have defined the PstA family as a conserved and specific c-di-AMP receptor in bacteria.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Dinucleoside Phosphates/metabolism , Signal Transduction , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Carrier Proteins/metabolism , Conserved Sequence , Humans , Hydrogen Bonding , Listeria monocytogenes/genetics , Listeria monocytogenes/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Conformation , Sequence Alignment
11.
Cell ; 158(6): 1389-1401, 2014 Sep 11.
Article in English | MEDLINE | ID: mdl-25215494

ABSTRACT

Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP-interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected catalytic activity of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic dinucleotide.


Subject(s)
Dinucleoside Phosphates/metabolism , Listeria monocytogenes/metabolism , Pyruvate Carboxylase/chemistry , Pyruvate Carboxylase/metabolism , Allosteric Regulation , Amino Acid Sequence , Animals , Bacteriolysis , Binding Sites , Crystallography, X-Ray , Host-Pathogen Interactions , Listeria monocytogenes/enzymology , Listeria monocytogenes/growth & development , Listeriosis/microbiology , Mice , Models, Molecular , Molecular Sequence Data
12.
Biochemistry ; 52(3): 488-96, 2013 Jan 22.
Article in English | MEDLINE | ID: mdl-23286247

ABSTRACT

Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO2 donor. Studies with Escherichia coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of the tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations into the BC domain dimer interface of Staphylococcus aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A, and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/metabolism , Pyruvate Carboxylase/chemistry , Pyruvate Carboxylase/metabolism , Staphylococcus aureus/enzymology , Amino Acid Substitution , Bacterial Proteins/genetics , Biocatalysis , Carbon-Nitrogen Ligases/genetics , Catalytic Domain , Chromatography, Gel , Crystallography, X-Ray , Holoenzymes/chemistry , Holoenzymes/genetics , Holoenzymes/metabolism , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Conformation , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Pyruvate Carboxylase/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ultracentrifugation
13.
Biopolymers ; 96(5): 586-95, 2011.
Article in English | MEDLINE | ID: mdl-22180906

ABSTRACT

The design and synthesis of protein-like polymers is a fundamental challenge in materials science. A means to achieve this goal is to create synthetic polymers of defined sequence where all relevant folding information is incorporated into a single polymer strand. We present here the aqueous self-assembly of peptoid polymers (N-substituted glycines) into ultrathin, two-dimensional highly ordered nanosheets, where all folding information is encoded into a single chain. The sequence designs enforce a two-fold amphiphilic periodicity. Two sequences were considered: one with charged residues alternately positive and negative (alternating patterning), and one with charges segregated in positive and negative halves of the molecule (block patterning). Sheets form between pH 5 and 10 with the optimal conditions being pH 6 for the alternating sequence and pH 8 for the block sequence. Once assembled, the nanosheets remain stable between pH 6 and 10 with observed degradation beginning to occur below pH 6. The alternating charge nanosheets remain stable up to concentrations of 20% acetonitrile, whereas the block pattern displayed greater robustness remaining stable up to 30% acetonitrile. These observations are consistent with expectations based on considerations of the molecules' electrostatic interactions. This study represents an important step in the construction of abiotic materials founded on biological informatic and folding principles.


Subject(s)
Nanotechnology , Peptidomimetics , Peptoids/chemistry , Acetonitriles/chemistry , Computer Simulation , Hydrogen-Ion Concentration , Microscopy, Electron, Scanning , Molecular Structure , Protein Folding , Protein Structure, Secondary , Static Electricity
14.
Nat Mater ; 9(5): 454-60, 2010 May.
Article in English | MEDLINE | ID: mdl-20383129

ABSTRACT

The design and synthesis of protein-like polymers is a fundamental challenge in materials science. A biomimetic approach is to explore the impact of monomer sequence on non-natural polymer structure and function. We present the aqueous self-assembly of two peptoid polymers into extremely thin two-dimensional (2D) crystalline sheets directed by periodic amphiphilicity, electrostatic recognition and aromatic interactions. Peptoids are sequence-specific, oligo-N-substituted glycine polymers designed to mimic the structure and functionality of proteins. Mixing a 1:1 ratio of two oppositely charged peptoid 36mers of a specific sequence in aqueous solution results in the formation of giant, free-floating sheets with only 2.7 nm thickness. Direct visualization of aligned individual peptoid chains in the sheet structure was achieved using aberration-corrected transmission electron microscopy. Specific binding of a protein to ligand-functionalized sheets was also demonstrated. The synthetic flexibility and biocompatibility of peptoids provide a flexible and robust platform for integrating functionality into defined 2D nanostructures.


Subject(s)
Biomimetics , Glycine/analogs & derivatives , Peptoids/chemistry , Polymers/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Buffers , Crystallization , Fourier Analysis , Ligands , Models, Chemical , Nanostructures/chemistry , Nanostructures/ultrastructure , Polymers/chemical synthesis , Protein Binding , Sequence Homology, Amino Acid , Static Electricity , Water/chemistry
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