Thermodynamic aspects of the CO-binding reaction to cytochrome P-450cam. Relevance with their biological significance and structure

The CO-binding kinetics of cytochrome P-450_cam(+) and P-450cam(−) have been measured in the millisecond time domain using a flash photolysis method. We have determined the reaction coordinates for free energy, enthalpy and entropy from the temperature dependence of the overall rate constants of the bimolecular forward (on) and backward (off) reactions. Comparing the thermodynamic profiles of P-450_cam with that of myoglobin (Mb) reported so far, the enthalpy and the entropy coordinates exhibit the following remarkable characteristics. The CO-binding equilibrium: The stability of the CO-complex is perfectly entropy-driven for P-450_cam, while enthalpy-driven for Mb. This entropy-driven feature for P-450_cam is enhanced by the dissociating d-camphor. The on and off activation processes: The on and off reactions for P-450_cam.are dominantly controlled by the enthalpy and entropy terms, respectively, while those for Mb are rather the reverse of the case of P-450_cam. The dissociation of d-camphor has a significant effect on the on reaction but no effect on the off reaction. Analyzing these thermodynamic features on the basis of the physical chemistry in the solution reaction, it was found that these characteristic profiles arise from the difference in the global structural change between the proteins. Namely, during the equilibrium process of the CO binding, this structural change is accompanied by a larger increase in the degree of freedom in P-450_cam than in Mb. We discussed the correlations between the structural changes and their biological significance.     

Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae

We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called “inhibitor cystine knot” or “knottin” fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a “membrane access” mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.     

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin,a coiled-coil protein,in Caenorhabditis elegans muscle

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a K_D of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.     

Identification of SPOR Domain Amino Acids Important for Septal Localization,Peptidoglycan Binding,and a Disulfide Bond in the Cell Division Protein FtsN

SPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein (^TTGFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a β-sheet in the published solution structure of FtsN^SPOR. Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share <20% amino acid identity. Taken together, these findings support the proposal that SPOR domains localize by binding to septal peptidoglycan and imply that the binding site is associated with the β-sheet. We also show that FtsN^SPOR contains a disulfide bond between β-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.     

Coexistence of Flexibility and Stability of Proteins: An Equation of State

We consider a recently suggested “equation of state” for natively folded proteins, and verify its validity for a set of about 5800 proteins. The equation is based on a fractal viewpoint of proteins, on a generalization of the Landau-Peierls instability, and on a marginal stability criterion. The latter allows for coexistence of stability and flexibility of proteins, which is required for their proper function. The equation of state relates the protein fractal dimension , its spectral dimension , and the number of amino acids N. Using structural data from the protein data bank (PDB) and the Gaussian network model (GNM), we compute and for the entire set and demonstrate that the equation of state is well obeyed. Addressing the fractal properties and making use of the equation of state may help to engineer biologically inspired catalysts.     

Crystal structure of a trimeric form of the KV7.1 (KCNQ1) A‐domain tail coiled‐coil reveals structural plasticity and context dependent changes in a putative coiled‐coil trimerization motif

Coiled-coils are widespread protein–protein interaction motifs typified by the heptad repeat (abcdefg)_n in which “a” and “d” positions are hydrophobic residues. Although identification of likely coiled-coil sequences is robust, prediction of strand order remains elusive. We present the X-ray crystal structure of a short form (residues 583–611), “Q1-short,” of the coiled-coil assembly specificity domain from the voltage-gated potassium channel Kv7.1 (KCNQ1) determined at 1.7 Å resolution. Q1-short lacks one and half heptads present in a previously studied tetrameric coiled-coil construct, Kv7.1 585–621, “Q1-long.” Surprisingly, Q1-short crystallizes as a trimer. In solution, Q1-short self-assembles more poorly than Q1-long and depends on an R-h-x-x-h-E motif common to trimeric coiled-coils. Addition of native sequences that include “a” and “d” positions C-terminal to Q1-short overrides the R-h-x-x-h-E motif influence and changes assembly state from a weakly associated trimer to a strongly associated tetramer. These data provide a striking example of a naturally occurring amino sequence that exhibits context-dependent folding into different oligomerization states, a three-stranded versus a four-stranded coiled-coil. The results emphasize the degenerate nature of coiled-coil energy landscapes in which small changes can have drastic effects on oligomerization. Discovery of these properties in an ion channel assembly domain and prevalence of the R-h-x-x-h-E motif in coiled-coil assembly domains of a number of different channels that are thought to function as tetrameric assemblies raises the possibility that such sequence features may be important for facilitating the assembly of intermediates en route to the final native state.     

Molecular interactions involved in proton-dependent gating in KcsA potassium channels

The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel’s response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod–Wyman–Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a “strong” sensor displaying a large shift in pK_a between closed and open states, and E118 is a “weak” pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pK_a values of the pH-sensing residues was sufficient to capture the effects of all mutations.     

Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The “R-helix” of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the “R-helix linker”. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-LIKE gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.Key words: ClC channel, chloride channel, homology model     

Structural basis for two-component system inhibition and pilus sensing by the auxiliary CpxP protein

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in Gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like “left hands” forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.     

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys¹⁹-Pro²⁰-Trp²¹-Cys²² motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a “closed” compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an “open” conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.     

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little ¹⁴CO₂ was formed from the ¹⁴C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and “Methanococcus aeolicus” are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.     

Rare Branched Fatty Acids Characterize the Lipid Composition of the Intra-Aerobic Methane Oxidizer “Candidatus Methylomirabilis oxyfera”

The recently described bacterium “Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “Ca. Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “Ca. Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “Ca. Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC_16:0). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC_16:1Δ7), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC_16:0 and 10MeC_16:1Δ7 are key and characteristic components of the lipid profile of “Ca. Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.     

Deletion of Genes Encoding Arginase Improves Use of “Heavy” Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when ¹³C₆-arginine (Arg-6) is used for labeling, it is less successful when ¹³C₆ ¹⁵N₄-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of ¹³C₅ ¹⁵N₂-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.     

Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Δ290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Δ290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 × 10⁻⁸ M versus 3.2 × 10⁻⁶ M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Δ290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster k_on rather than to a slower k_off. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 × 10⁻⁵ M) than did gD1(306t) due to a more rapid k_off. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

Cytochrome Involvement in Mn(II) Oxidation by Two Marine Bacteria

Two marine, Mn(II)-oxidizing bacterial cultures, BIII 45 and BIII 82, were examined spectrophotometrically at ambient temperature for their cytochrome complements. Membrane preparations from an ethylenediaminetetracetate-lysozyme treatment of 48-h cultures of both strains contained type b, c, and o cytochromes. No evidence for a type a cytochrome was noted. “Periplasmic” fractions of both strains also contained small amounts of cytochrome, including cytochrome o, but “intracellular” fractions did not. Type c cytochrome in membrane preparations of culture BIII 45 was consistently reduced by Mn(II) when the membranes were suspended in the periplasmic fraction of the culture. In the case of culture BIII 82, type c cytochrome in membrane preparations was consistently reduced by Mn(II) when the membranes were suspended in either periplasmic or intracellular fractions of the strain. Although, based on previous inhibitor studies, type b cytochrome was also expected to be reduced by Mn(II), no spectrophotometric evidence for its reduction was found, probably because not enough of it was reduced under the steady-state conditions of the experiments.     

The yoga of Rag GTPases: Dynamic structural poses confer amino acid sensing by mTORC1

Heterodimeric Rag GTPases play a critical role in relaying fluctuating levels of cellular amino acids to the sensor mechanistic target of rapamycin complex 1. Important mechanistic questions remain unresolved, however, regarding how guanine nucleotide binding enables Rag GTPases to transition dynamically between distinct yoga-like structural poses that control activation state. Egri and Shen identified a critical interdomain hydrogen bond within RagA and RagC that stabilizes their GDP-bound states. They demonstrate that this long-distance interaction controls Rag structure and function to confer appropriate amino acid sensing by mechanistic target of rapamycin complex 1.

Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse cellular cues to promote cell growth and proliferation (1, 2). Sufficient levels of nutrients such as amino acids are required for growth factors and hormones (e.g., IGF-1 and insulin) to activate mTORC1 via PI3K, Akt, Ras homolog enriched in the brain (Rheb) (a small GTPase), and tuberous sclerosis complex (a GTPase-activating protein for Rheb) (Fig. 1A). mTORC1 signaling in turn drives anabolic (e.g., protein synthesis) and suppresses catabolic (e.g., autophagy) cellular processes. Evolutionarily conserved Rag GTPases play a critical role in amino acid sensing by mTORC1 (3, 4). Despite advances in understanding Rag structure and function, important mechanistic questions remain regarding how dynamic structural states of Rag proteins controlled by guanine nucleotide binding confer amino acid sensing by mTORC1. Egri and Shen used elegant kinetic and cell-based methods to quantitatively dissect dynamic structural elements within Rag subunits that enable mTORC1 to respond to fluctuating levels of amino acids appropriately and rapidly (5).Open in a separate windowFigure 1mTORC1 activation by growth factors (GFs) requires sufficient levels of amino acids (AAs). GFs and hormones (e.g., IGF-1; insulin) signal through PI3K, Akt, and TSC and activate Rheb through increased GTP loading (A). AAs drive Rag heterodimers toward a RagA/B^GTP–RagC/D^GDP “on” state; conversely, AA deprivation induces a switch toward a RagA/B^GDP–RagC/D^GTP “off” state. In the “on” state, Rag heterodimers bind to and recruit mTORC1 to the surface of lysosomes, where Rheb resides. Therefore, AAs and GFs activate mTORC1 cooperatively because of an induced proximity mechanism mediated by Rags and Rheb. A critical hydrogen bond (blue bar) between the NBD and CRD of RagA or RagC plays a critical role in maintaining the two stable “on” and “off” states (B). CRD, C-terminal roadblock domain; mTORC1, mechanistic target of rapamycin complex 1; NBD, nucleotide-binding domain; Rheb, Ras homolog enriched in the brain; TSC, tuberous sclerosis complex.Rag proteins function as obligate heterodimers, whereby mammalian RagA or RagB dimerizes with RagC or RagD. Rag proteins localize to lysosomal membranes by tethering to the LAMTOR/Ragulator complex (Fig. 1A) (6). In the active RagA/B^GTP–RagC/D^GDP state formed in amino acid–replete conditions, the Rag heterodimer recruits mTORC1 to the lysosomal surface through direct binding (6). Such recruitment enables Rheb to associate with and activate mTORC1 by an induced proximity mechanism (7). Upon amino acid withdrawal, GTP on RagA/B hydrolyzes to GDP, and GTP exchanges for GDP on RagC/D. This inactive RagA/B^GDP–RagC/D^GTP heterodimer releases mTORC1 into the cytosol. Thus, Rags function as dynamic molecular switches that control mTORC1 signaling in accordance with amino acid levels.Prior work (8) demonstrated that the two GTPase subunits of the Rag heterodimer (RagA/B and RagC/D) communicate with each other. GTP binding to one subunit limits binding of GTP to the other subunit and increases GTP hydrolysis if binding were to occur, and vice versa. Such intersubunit crosstalk prevents dual GTP loading, thus maintaining an opposite guanine nucleotide–loaded state and driving Rag heterodimers into two stable “on” or “off” states. The crystal structure of Rag heterodimers from budding yeast bound to GDP or GTP provided important structural information regarding how guanine nucleotide binding controls Rag architecture (9, 10). An individual Rag subunit consists of a nucleotide-binding domain (NBD) and a C-terminal roadblock domain (CRD) that mediates heterodimerization. In the GDP-bound state, the switch I domain within the NBD forms an alpha helix that orients toward the CRD; in the GTP-bound state, the switch I domain swings upward to the top of the nucleotide-binding pocket, away from the CRD. From the yeast Rag crystal structures (9, 10), Egri and Shen predicted that in the GDP- but not GTP-bound state, the hydroxyl group of Ser266 in the RagC CRB forms hydrogen bonds with Lys84 in the switch I alpha helix of the RagC NBD. As RagA Thr210 is analogous to RagC Ser266, they also predicted that Thr210 in the RagA CRB forms hydrogen bonds with Asn30 in the NBD. In the GTP-bound state, the switch I domain swings up and away from the CRD, preventing formation of these hydrogen bonds (Fig. 1B).Egri and Shen coupled these predictions with elegant quantitative kinetic in vitro assays of guanine nucleotide loading and GTP hydrolysis to demonstrate that a critical interdomain interaction in RagA and RagC maintains an opposite nucleotide-loading state in heterodimers and regulates mTORC1 activity (5). They first mutated RagA Thr210 and RagC Ser266 to Ala to abrogate the hydrogen bond and then biochemically purified WT and mutant Rag heterodimers. Ablation of the hydrogen bond had no effect on guanine nucleotide binding. When only one GTP was bound to the heterodimer, rates of GTP hydrolysis were similar on WT and mutant Rag heterodimers. When both Rag subunits of the heterodimer were forced to bind GTP, WT heterodimers displayed an increased rate of GTP hydrolysis compared with those loaded with a single GTP, indicating that the heterodimer actively resolves the dual GTP problem by hydrolyzing GTP on one subunit, consistent with prior work (8). GTP hydrolysis was increased even more for the RagA(T210A)–RagC and RagA–RagC(S266A) mutant heterodimers, suggesting that the mutations mimic a constitutive GTP-loaded conformation, driving faster GTP hydrolysis on the other subunit. In WT heterodimers, preloading the first subunit with GTP increased GTP hydrolysis on the other subunit relative to preloading with GDP. Interestingly, radioactive GTP hydrolysis in mutant heterodimers was strikingly faster than that of the WT when preloaded with either GTP or GDP, indicating that the RagA(T210) and RagC(S266A) mutations shift the heterodimer toward the GTP-loaded conformation. These results suggest that the hydrogen bond stabilizes the GDP-loaded state, and in its absence, Rag proteins tend to adopt a GTP-bound conformation even when bound to GDP, which accelerates GTP hydrolysis on the other subunit.Egri and Shen also investigated the functional significance of the RagA and RagC hydrogen bond in the control of mTORC1 signaling. Coimmunoprecipitation experiments and analysis of mTORC1 signaling to its well-established substrate S6K1 in intact cells demonstrated that the RagA(T210A)–RagC mutant associated with and activated mTORC1 inappropriately in the absence of amino acids. Upon amino acid stimulation, the RagA–RagC(S266A) mutant displayed reduced mTORC1 binding and failed to activate mTORC1 signaling. These results are consistent with RagA(T210A) mimicking a RagA^GTP “on” state and RagC(S266A) mimicking a RagC^GTP “off” state. Taken together, these results reveal the functional significance of the RagA and RagC interdomain hydrogen bond, demonstrating that it plays a critical role in regulation of mTORC1 signaling in accordance with amino acid levels.Mechanistic understanding of Rag heterodimer asanas (i.e., postures and poses) will improve our understanding of the role of mTORC1 in tumorigenesis and metabolism. For example, cancer-associated mutations have been identified in RagC, which increase mTORC1 binding (2). In addition, the physiologic importance of Rag proteins in metabolic control was demonstrated in mice engineered with an active RagA knock-in allele conferring constitutive GTP loading. These mice die perinatally, as they are unable to suppress mTORC1 signaling appropriately upon severance of the placental nutrient supply at birth. These mice fail to suppress energy expenditure, fail to induce autophagy and liberate amino acids as substrates for gluconeogenesis, and consequently fail to upregulate hepatic glucose production, responses essential for survival during fasting, unlike WT neonates (2). Thus, Rag GTPases play critical roles in cell and organismal physiology. Moving forward, deeper mechanistic insight into the yoga of Rag GTPases will improve our understanding of nutrient sensing, how its aberrant regulation contributes to a host of diseases such as cancer, obesity, and type II diabetes, and how its therapeutic targeting could treat these disorders. Namaste.