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ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3 which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and non-productively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and non-productive binding by the incorrect substrate bears strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from non-linearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These non-specific interactions likely act to lower the available intra-cellular GTP and ATP concentrations and may have driven evolution to adapt the Michaelis constants of NMP kinases accordingly.

Vitiligo is an epidermal disorder causes depigmented patches resulted from the loss of melanocytes, Autoimmunity hypotheses strongly supports that the immune system compartments responsible in the development of vitiligo. Adenosine MonoPhosphate kinase (AMPK) signaling plays a role in regimentation in vitiligo. In this present study, set of ligands selected to dock against AMPK protein in the AMP binding site using FlexX software. Based on the scores and protein-ligand interactions selected ligands were analyzed for its binding affinity and protein ligand stability for its further drug development process.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

Iron (Fe) (oxyhydr)oxide minerals, which are amongst most reactive minerals in soils and sediments, are known to exhibit strong adsorption of inorganic phosphate (Pi) and organophosphate (Po) compounds. Beyond synthetic Po compounds, much still remains unknown about the reactivity of these minerals to transform naturally-occurring Po compounds to Pi, particularly with respect to solution versus surface speciation of Po hydrolysis. To investigate this reactivity with a ferrihydrite-type mineral and ribonucleotides, we employed high-resolution liquid chromatography-mass spectrometry (LC-MS), X-ray absorption near-edge structure (XANES), Fourier-transform infrared (FTIR) spectroscopy, and molecular modeling. Kinetic experiments were conducted with the mineral (1 g L−1) reacted with adenosine monophosphate, diphosphate, or triphosphate (respectively AMP, ADP, ATP; 50 µM). Analysis of solution organic species by LC-MS implied that only adsorption occurred with AMP and ADP but both adsorption and dephosphorylation of ATP were evident. Maximum adsorption capacities per gram of mineral were 40.6 ± 0.8 µmol AMP, 35.7 ± 1.6 µmol ADP, and 10.9 ± 1.0 µmol ATP; solution dephosphorylated by-products accounted for 15% of initial ATP. Subsequent XANES analysis of the surface species revealed that 16% of adsorbed AMP and 30% of adsorbed ATP were subjected to dephosphorylation, which was not fully quantifiable from the solution measurements. Molecular simulations predicted that ADP and ATP were complexed mainly via the phosphate groups whereas AMP binding also involved multiple hydrogen bonds with the adenosine moiety; our FTIR data confirmed these binding confirmations. Our findings thus imply that specific adsorption mechanisms dictate the recycling and subsequent trapping of Pi from ribonucleotide-like biomolecules reacted with Fe (oxyhydr)oxide minerals.

Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible spectrum of light, but the regulation of the chromophorylation of CBCRs is unknown. Slr1393 from Synechocystis sp. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. The protein Slr2111 from Synechocystis sp. PCC 6803 includes a cystathionine β-synthase (CBS) domain pair of an as yet unknown function at its N terminus. CBS domains are often characterized as sensors of cellular energy status by binding nucleotides. In this work, we demonstrate that Slr2111 strongly interacts with Slr1393 in vivo and in vitro, which generates a complex in a 1:1 molar ratio. This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. Instead, the complex stability and thereby the chromophorylation of Slr1393 are regulated by the binding of nucleotides (ATP, ADP, AMP) to the CBS domains of Slr2111 with varying affinities. It is demonstrated that residues Asp-53 and Arg-97 of Slr2111 are involved in nucleotide binding. While ATP binds to Slr2111, the association between the two proteins gets weaker and chromophorylation of Slr1393 are enabled. In contrast, AMP binding to Slr2111 leads to a stronger association, thereby inhibiting the chromophorylation. It is concluded that Slr2111 acts as a sensor of the cellular energy status that regulates the chromophorylation of Slr1393 and thereby its function as a light-driven histidine kinase.

PRKAG2 encodes the γ2-subunit isoform of 5′-AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in the regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload, ventricular preexcitation, and hypertrophy. We identified a novel, de novo PRKAG2 mutation (K475E) in a neonate with prenatal onset of HCM. We aimed to investigate the cellular impact, signaling pathways involved, and therapeutic options for K475E mutation using cells stably expressing human wild-type (WT) or the K475E mutant. In human embryonic kidney-293 cells, the K475E mutation induced a marked increase in the basal phosphorylation of T172 and AMPK activity, reduced sensitivity to AMP in allosteric activation, and a loss of response to phenformin. In H9c2 cardiomyocytes, the K475E mutation induced inhibition of AMPK and reduced the response to phenformin and increases in the phosphorylation of p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Primary fibroblasts from the patient with the K475E mutation also showed marked increases in the phosphorylation of p70S6K and 4E-BP1 compared with those from age-matched, nondiseased controls. Moreover, overexpression of K475E induced hypertrophy in H9c2 cells, which was effectively reversed by treatment with rapamycin. Taken together, we have identified a novel, de novo infantile-onset PRKAG2 mutation causing HCM. Our study suggests the K475E mutation induces alteration in basal AMPK activity and results in a hypertrophy phenotype involving the mechanistic target of rapamycin signaling pathway, which can be reversed with rapamycin. NEW & NOTEWORTHY We identified a novel, de novo PRKAG2 mutation (K475E) in the cystathionine β-synthase 3 repeat, a region critical for AMP binding but with no previous reported mutation. Our data suggest the mutation affects AMP-activated protein kinase activity, activates cell growth pathways, and results in cardiac hypertrophy, which can be reversed with rapamycin.

Antimicrobial peptides (AMPs) can target bacterial membranes and kill bacteria through membrane structural damage and cytoplasmic leakage. A group of surfactant-like cationic AMPs was developed from substitutions to selective amino acids in the general formula of G(IIKK)3I-NH2, (called G3, a de novo AMP), to explore the correlation between AMP hydrophobicity and bioactivity. A threshold surface pressure over 12 mN/m was required to cause measurable antimicrobial activity and this corresponded to a critical AMP concentration. Greater surface activity exhibited stronger antimicrobial activity but had the drawback of worsening hemolytic activity. Small unilamellar vesicles (SUVs) with specific lipid compositions were used to model bacterial and host mammalian cell membranes by mimicking the main structural determinants of the charge and composition. Leakage from the SUVs of encapsulated carboxyfluorescein measured by fluorescence spectroscopy indicated a negative correlation between hydrophobicity and model membrane selectivity, consistent with measurements of the zeta potential that demonstrated the extent of AMP binding onto model SUV lipid bilayers. Experiments with model lipid membranes thus explained the trend of minimum inhibitory concentrations and selectivity measured from real cell systems and demonstrated the dominant influence of hydrophobicity. This work provides useful guidance for the improvement of the potency of AMPs via structural design, whilst taking due consideration of cytotoxicity.

Through transcriptional control of the evolutionarily conserved heat shock, or proteotoxic stress, response, heat shock factor 1 (HSF1) preserves proteomic stability. Here, we show that HSF1, a physiological substrate for AMP-activated protein kinase (AMPK), constitutively suppresses this central metabolic sensor. By physically evoking conformational switching of AMPK, HSF1 impairs AMP binding to the γ subunits and enhances the PP2A-mediated de-phosphorylation, but it impedes the LKB1-mediated phosphorylation of Thr172, and retards ATP binding to the catalytic α subunits. These immediate and manifold regulations empower HSF1 to both repress AMPK under basal conditions and restrain its activation by diverse stimuli, thereby promoting lipogenesis, cholesterol synthesis, and protein cholesteroylation. In vivo, HSF1 antagonizes AMPK to control body fat mass and drive the lipogenic phenotype and growth of melanomas independently of its intrinsic transcriptional action. Thus, the physical AMPK-HSF1 interaction epitomizes a reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine.

Antimicrobial Peptides (AMPs) are host defense molecules that initiate microbial death by binding to the membrane. On membrane binding, AMPs undergo changes in conformation and aggregation state to enable killing action. Depending on the AMP and cell membrane characteristics, the nature of binding can be aggregating or non-aggregating, with high/low cooperativity, at single or multiple sites with high/low affinity leading to a unique killing action that needs to be studied individually. In the present study, a steady-state model that simulates AMP-membrane interaction was developed and was used to predict the mechanism of AMP binding. The predictions obtained from the model were validated with experimentally deciphered values available in literature. The model was further used to predict the mechanism for a set of designed AMPs with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family. Depending on the predicted mechanism, a unique half saturation constant and steepness of response (Hill coefficient) was obtained which was further validated with available data from literature. The model could reliably predict the mechanism, the half saturation constant and the Hill coefficient values. Further based on the analysis, it was observed that aggregation and oligomerization result in drastic killing action in a short range of peptide concentration owing to high Hill coefficient values. Mechanisms such as monomers binding at multiple sites with/without cooperativity result in antimicrobial activity at low half saturation constant though the killing action may not be steep. Thus, the methodology developed here can be used to develop hypothesis for studying AMP-membrane interaction mechanisms.

Fatty acid transporter proteins (FATPs) are a family (SLC27) of six transporters (FATP1-6). They have at least one, and possibly six [4, 11], transmembrane segments, and are predicted on the basis of structural similarities to form dimers. SLC27 members have several structural domains: integral membrane associated domain, peripheral membrane associated domain, FATP signature, intracellular AMP binding motif, dimerization domain, lipocalin motif, and an ER localization domain (identified in FATP4 only) [2, 8, 9]. These transporters are unusual in that they appear to express intrinsic very long-chain acyl-CoA synthetase (EC 6.2.1.- , EC 6.2.1.7) enzyme activity. Within the cell, these transporters may associate with plasma and peroxisomal membranes. FATP1-4 and -6 transport long- and very long-chain fatty acids, while FATP5 transports long-chain fatty acids as well as bile acids [7, 11].

Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis. It is a potential drug target in the treatment of type II diabetes. The protein is also associated with a rare inherited metabolic disease and some cancer cells lack FBPase activity which promotes glycolysis facilitating the Warburg effect. Thus, there is interest in both inhibiting the enzyme (for diabetes treatment) and restoring its activity (in relevant cancers). The mammalian enzyme is tetrameric, competitively inhibited by Fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the active site of the enzyme. A recent paper by Topaz et al. (Bioscience Reports (2019) 39, pii:BSR20180960) has added additional detail to our understanding of this information transmission process. Two residues in the AMP binding site (Lys112 and Tyr113) were shown to be involved in initiating the message between the two sites. This tyrosine residue has recently be shown to be important with protein’s interaction with the antidiabetic drug metformin. A variant designed to increase metal ion affinity (M248D) resulted in a five-fold increase in enzymatic activity. Interestingly alterations of two residues at the subunit interfaces (Tyr164 and Met177) resulted in increased responsiveness to AMP. Overall, these findings may have implications in the design of novel FBPase inhibitors or activators.

Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.

When crystallized in the absence of the allosteric inhibitor AMP, human muscle fructose-1,6-bisphosphatase has a totally unexpected quaternary structure of its active R form, with the two dimers of the homotetrameric molecule in a perpendicular orientation, in stark contrast to the coplanar arrangement of the closely related liver isozyme. The T-to-R switch of the muscle enzyme also involves a highly unusual α→β refolding of the N-terminus., Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and is a key enzyme of gluconeogenesis and glyconeogenesis and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrates, and notably Homo sapiens, express two FBPase isoforms. The liver isozyme is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis. The muscle isoform is expressed in all cells, and recent studies have demonstrated that its role goes far beyond the enzymatic function, as it can interact with various nuclear and mitochondrial proteins. Even in its enzymatic function, the muscle enzyme is different from the liver isoform, as it is 100-fold more susceptible to allosteric inhibition by AMP and this effect can be abrogated by complex formation with aldolase. All FBPases are homotetramers composed of two intimate dimers: the upper dimer and the lower dimer. They oscillate between two conformational states: the inactive T form when in complex with AMP, and the active R form. Parenthetically, it is noted that bacterial FBPases behave somewhat differently, and in the absence of allosteric activators exist in a tetramer–dimer equilibrium even at relatively high concentrations. [Hines et al. (2007 ▸), J. Biol. Chem. 282, 11696–11704]. The T-to-R transition is correlated with the conformation of the key loop L2, which in the T form becomes ‘disengaged’ and unable to participate in the catalytic mechanism. The T states of both isoforms are very similar, with a small twist of the upper dimer relative to the lower dimer. It is shown that at variance with the well studied R form of the liver enzyme, which is flat, the R form of the muscle enzyme is diametrically different, with a perpendicular orientation of the upper and lower dimers. The crystal structure of the muscle-isozyme R form shows that in this arrangement of the tetramer completely new protein surfaces are exposed that are most likely targets for the interactions with various cellular and enzymatic partners. The cruciform R structure is stabilized by a novel ‘leucine lock’, which prevents the key residue, Asp187, from locking loop L2 in the disengaged conformation. In addition, the crystal structures of muscle FBPase in the T conformation with and without AMP strongly suggest that the T-to-R transition is a discrete jump rather than a shift of an equilibrium smooth transition through multiple intermediate states. Finally, using snapshots from three crystal structures of human muscle FBPase, it is conclusively demonstrated that the AMP-binding event is correlated with a β→α transition at the N-terminus of the protein and with the formation of a new helical structure.

CshA is a DEAD-box RNA helicase that belongs to the DExD/H-box family of proteins, which generally have an RNA-dependent ATPase activity. In Staphylococcus aureus, CshA was identified as a component of the RNA degradosome and plays important roles in RNA turnover. In this study, the crystal structures of the N-terminal RecA-like domain 1 of S. aureus CshA (SaCshAR1) and of its complex with AMP (SaCshAR1–AMP) are reported at resolutions of 1.5 and 1.8 Å, respectively. SaCshAR1 adopts a conserved α/β RecA-like structure with seven parallel strands surrounded by nine α-helices. The Q motif and motif I are responsible for the binding of the adenine group and phosphate group of AMP, respectively. Structure comparison of SaCshAR1–AMP and SaCshAR1 reveals that motif I undergoes a conformational change upon AMP binding. Isothermal titration calorimetry assays further conformed the essential roles of Phe22 in the Q motif and Lys52 in motif I for binding ATP, indicating a conserved substrate-binding mechanism in SaCshA compared with other DEAD-box RNA helicases.

Background: PstA is a cyclic di-AMP receptor and PII-like signal transduction protein. Results: Apo-PstA and complex crystal structures reveal a novel cyclic di-AMP-binding mode and induced conformational changes. Conclusion: PstA has a similar fold but distinct signal transduction properties from classic PII proteins, which function in nitrogen metabolism. Significance: Identification of common features allows for rational prediction of cyclic di-AMP-binding sites., Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3′3′-cGAMP) but not c-di-GMP or 2′3′-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA. Background: Cyclic di-AMP is an essential second messenger in eubacteria. Results: The c-di-AMP receptor DarA was identified in B. subtilis. The crystal structure and ITC data revealed the nucleotide specificity of DarA. Conclusion: DarA is a PII-like protein that undergoes conformational changes upon c-di-AMP binding. Significance: A novel PII-like protein is involved in c-di-AMP signaling.

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.

Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections. However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae , a Gram-positive pathogen. In this study, we identified a c -di- A MP b inding p rotein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal potassium uptake.

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg(2+) except for the C. novyi PPase where Mg(2+) produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.

One fundamental issue in the field of AMPs has been whether the action of AMPs produces pores in the membranes or disintegrates the membranes by the saturated surface binding of AMPs (the carpet model). In the absence of clear evidence, either mechanism seems to explain the phenomena of bacteria lysis. Even if the issue were resolved in the model membrane studies, it is not clear if the result of model membranes extends to bacterial membranes. Here we investigated the action of AMPs on E. coli spheroplasts. The absence of the outer membrane made it possible to observe the action of AMPs on the cytoplasmic membranes. Previously we found that the properties of the bacterial cell membranes are dominated by a membrane reservoir, significantly different from that of a GUV. Furthermore, these unique properties of bacterial membranes are metabolically maintained. We would like to know how the actions of AMPs on spheroplast membranes compare with the actions on GUVs. We compared the actions of human AMP LL-37 and melittin on spheroplasts and GUVs. We developed a fluorescence recovery after photobleaching (FRAP) technique to examine the dye leakage through the bacterial membranes. AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the responses of GUVs. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of FRAP showed that the fluxes of dye molecules in and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. The effects of LL37 and melittin are qualitatively the same.

AMP-activated protein kinase (AMPK), a serine/threonine kinase, is a highly conserved homeostatic regulatory enzyme with a plethora of important roles in energy storage and use. ATP is the primary cellular energy source; in situations of metabolic deficiency, its breakdown product AMP accumulates. AMPK is sensitive to the cellular energy state: it is activated by AMP and inactivated by ATP. Under metabolic stress, when the AMP/ATP ratio is elevated, AMPK acts as an energy sensor and compensates for energy depletion by upregulating energy sources and downregulating energy-consuming processes that are not immediately essential.1–5 There is growing recognition that AMPK is particularly important in the heart, which demands more energy on a continuous basis than most other organs.1,2 AMPK seems to be a critical regulator of cardiac energy status and may be a potential therapeutic target. In this article, we will review the literature regarding cardiac AMPK and discuss evidence for a potentially important and underappreciated role of AMPK in cardiac electrophysiology and arrhythmia generation. AMPK is a heterotrimeric enzyme with 1 catalytic (α) and 2 regulatory (β and γ) subunits (Figure 1). Thr-172 in the α-subunit is a crucial phosphorylation site that regulates AMPK function. The β-subunit has 2 structural components: a glycogen-binding domain that is sensitive to fuel storage levels and a C-terminal region that anchors α- and γ-subunits. The γ-subunit has a pair of cystathionine-β-synthase sequence repeats, called Bateman domains, which bind adenosine nucleotides.1–5 AMP binding to the γ-subunit facilitates Thr-172 phosphorylation by upstream kinases, protects the site against dephosphorylation by protein phosphatases (PPs), and allosterically activates the enzyme. The crystal structure of AMPK has recently been resolved, revealing how the regulatory domain stabilizes the activation loop of the kinase domain and prevents dephosphorylation.6 Various isoforms of the AMPK subunits have been identified …

This paper demonstrates and systematically characterizes the enrichment of biomolecular compounds using aptamer-functionalized surfaces within a microfluidic device. The device consists of a microchamber packed with aptamer-functionalized microbeads and integrated with a microheater and temperature sensor to enable thermally controlled binding and release of biomolecules by the aptamer. We first present an equilibrium binding-based analytical model to understand the enrichment process. The characteristics of the aptamer-analyte binding and enrichment are then experimentally studied, using adenosine monophosphate (AMP) and a specific RNA aptamer as a model system. The temporal process of AMP binding to the aptamer is found to be primarily determined by the aptamer-AMP binding kinetics. The temporal process of aptamer-AMP dissociation at varying temperatures is also obtained and observed to occur relatively rapidly (< 2 s). The specificity of the enrichment is next confirmed by performing selective enrichment of AMP from a sample containing biomolecular impurities. Finally, we investigate the enrichment of AMP by either discrete or continuous introduction of a dilute sample into the microchamber, demonstrating enrichment factors ranging from 566 to 686×, which agree with predictions of the analytical model.

Cytosolic fructose-1,6-bisphosphatase (cFBPase) in plants is a key regulatory enzyme in the photosynthetic sucrose biosynthesis. Plant cFBPases, like the mammalian FBPases, are inhibited by adenosine 5'-monophosphate (AMP) and fructose-2,6-bisphosphate (Fru-2,6-). In the mammalian FBPases, Lys112 and Tyr113 play important roles in the AMP binding. To understand roles of the corresponding residues, Lys115 and Tyr116, in pea cFBPase, the mutant cFBPases were generated by site-directed mutagenesis. The alterations of Lys115 to Gin and Tyr116 to Phe displayed small changes in and for Fru-2,6-, indicating that the mutation causes minor effects on the enzyme catalysis and Fru-2,6- binding, whereas resulted in higher than 500-fold increase of compared with that of the wild-type enzyme. Results indicate the residues Lys115 and Tyr116 play important roles in the binding of AMP to the allosteric site of the pea cFBPase.

Summary Staphylococcus aureus is a leading cause of hospital-associated and, more recently, community-associated infections caused by highly virulent methicillin-resistant strains (CA-MRSA). S. aureus survival in the human host is largely defined by the ability to evade attacks by antimicrobial peptides (AMPs) and other mechanisms of innate host defence. Here we show that AMPs induce resistance mechanisms in CA-MRSA via the aps AMP sensor/regulator system, including (i) the d-alanylation of teichoic acids, (ii) the incorporation of lysyl-phosphatidylglycerol in the bacterial membrane and a concomitant increase in lysine biosynthesis, and (iii) putative AMP transport systems such as the vraFG transporter, for which we demonstrate a function in AMP resistance. In contrast to the aps system of S. epidermidis, induction of the aps response in S. aureus was AMP-selective due to structural differences in the AMP binding loop of the ApsS sensor protein. Finally, using a murine infection model, we demonstrate the importance of the aps regulatory system in S. aureus infection. This study shows that while significant interspecies differences exist in the AMP–aps interaction, the AMP sensor system aps is functional and efficient in promoting resistance to a variety of AMPs in a clinically relevant strain of the important human pathogen S. aureus.

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. This crystal structure of the trimeric regulatory fragment of mammalian AMPK reveals the modes of AMP and ATP binding. AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment1. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner2. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways1,2. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the γ domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the γ domain bind either AMP or Mg·ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg·ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the α and/or β subunits binds to the γ subunit in the presence of AMP but not when ATP is bound.

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010, http://dx.doi.org/10.1074/jbc.M109.078725 ; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287: 418–428, 2012, http://dx.doi.org/10.1074/jbc.M111.290361 ). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli , possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.

Despite designating catalytic roles of Asp299 and Arg131 during the transfer of γ-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591–600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study, employing homology-model-guided site-specific protein mutagenesis, we show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in Km (Ado) with a 1000-fold decrease in kcat/Km, implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance of steric complementarity of the binding pocket. Use of 2′or 3′-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme–Ado complex, the binding energy (ΔΔGB) contribution of the former was greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding studies revealed that, unlike the R131A mutant, which showed abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered Km (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.

Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429–584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429–584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429–584 forms parallel dimers, commonly referred to as CBS (cystathionine β-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes ‘locked’ in its flat form.

In type 2 diabetes, the liver produces excessive amounts of glucose through the gluconeogenesis (GNG) pathway and consequently is partly responsible for the elevated glucose levels characteristic of the disease. In an effort to find safe and efficacious GNG inhibitors, we targeted the AMP binding site of fructose 1,6-bisphosphatase (FBPase). The hydrophilic nature of AMP binding sites and their widespread use for allosteric regulation of enzymes in metabolic pathways has historically made discovery of AMP mimetics suitable for drug development difficult. By using a structure-based drug design strategy, we discovered a series of compounds that mimic AMP but bear little structural resemblance. The lead compound, MB05032, exhibited high potency and specificity for human FBPase. Oral delivery of MB05032 was achieved by using the bisamidate prodrug MB06322 (CS-917), which is converted to MB05032 in two steps through the action of an esterase and a phosphoramidase. MB06322 inhibited glucose production from a variety of GNG substrates in rat hepatocytes and from bicarbonate in male Zucker diabetic fatty rats. Analysis of liver GNG pathway intermediates confirmed FBPase as the site of action. Oral administration of MB06322 to Zucker diabetic fatty rats led to a dose-dependent decrease in plasma glucose levels independent of insulin levels and nutritional status. Glucose lowering occurred without signs of hypoglycemia or significant elevations in plasma lactate or triglyceride levels. The findings suggest that potent and specific FBPase inhibitors represent a drug class with potential to treat type 2 diabetes through inhibition of GNG.

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated octameric enzyme composed of two types of homologous subunits designated IDH1 and IDH2. Based on sequence comparisons and structural models, both subunits are predicted to have adenine nucleotide binding sites. This was tested by alanine replacement of residues in putative sites in each subunit. Targets included adjacent aspartate/isoleucine residues implicated as important for determining cofactor specificity in related dehydrogenases and a residue in each IDH subunit in a position occupied by histidine in other cofactor binding sites. The primary kinetic effects of D286A/I287A and of H281A replacements in IDH2 were found to be a dramatic reduction in apparent affinity of the holoenzyme for NAD(+) and a concomitant reduction in V(max). Ligand binding assays also showed that the H281A mutant enzyme fails to bind NAD(+) under conditions that are saturating for the wild-type enzyme. In contrast, the primary effect of corresponding D279A/D280A and of R274A replacements in IDH1 is a reduction in holoenzyme binding of AMP, with concomitant alterations in kinetic and isocitrate binding properties normally associated with activation by this allosteric effector. These results suggest that the nucleotide cofactor binding site is primarily contributed by the IDH2 subunit, whereas the homologous nucleotide binding site in IDH1 has evolved for regulatory binding of AMP. These results are consistent with previous studies demonstrating that the catalytic isocitrate binding sites are comprised of residues primarily contributed by IDH2, whereas sites for regulatory binding of isocitrate are contributed by analogous residues of IDH1. In this study, we also demonstrate that a prerequisite for holoenzyme binding of NAD(+) is binding of isocitrate/Mg(2+) at the IDH2 catalytic site. This is comparable to the dependence of AMP binding upon binding of isocitrate at the IDH1 regulatory site.

Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling.

Fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis, is subject to metabolic regulation. The human muscle isozyme is significantly more sensitive towards the allosteric inhibitor, AMP, than the liver isoform. Here we report crystal structures and kinetic studies for wild-type human muscle Fru-1,6-Pase, the AMP-bound (1.6 Å), and product-bound complexes of the Q32R mutant, which was firstly introduced by an error in the cloning. Our high-resolution structure reveals for the first time that the higher sensitivity of the muscle isozyme towards AMP originates from an additional water-mediated, H-bonded network established between AMP and the binding pocket. Also present in our structures are a metaphosphate molecule, alternate conformations of Glu97 coordinating Mg(2+), and possible metal migration during catalysis. Although the individual subunit is similar to previously reported Fru-1,6-Pase structures, the tetrameric assembly of all these structures deviates from the canonical R- or T-states, representing novel tetrameric assemblies. Intriguingly, the concentration of AMP required for 50% inhibition of the Q32R mutant is increased 19-fold, and the cooperativity of both AMP and Mg(2+) is abolished or decreased. These structures demonstrate the Q32R mutation affects the conformations of both N-terminal residues and the dynamic loop 52-72. Also importantly, structural comparison indicates that this mutation in helix α2 is detrimental to the R-to-T conversion as evidenced by the absence of quaternary structural changes upon AMP binding, providing direct evidence for the critical role of helix α2 in the allosteric signal transduction.

Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma2 and gamma3. Both gamma2 (569 amino acids) and gamma3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma1. As with gamma1, mRNA encoding gamma2 is widely expressed in human tissues, whereas significant expression of gamma3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different gamma isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the gamma1 isoform, with the remaining activity being accounted for mainly by the gamma2 isoform. Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no evidence for any selective association between the alpha1 and alpha2isoforms and the various gamma isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the gamma isoform present, with gamma2-containing complexes having the greatest AMP-dependence, gamma3 the lowest, and gamma1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[(32)P]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14ω, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14ω, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5′ isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5′-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14ω.

MgADP - reacted with the nitrogenase molybdenum–iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31 P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8- 14 C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP - resulted in further AMP binding at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP 2- during reductant-limited turnover of nitrogenase under N 2 , indicating reversibility of ATP hydrolysis at 15 °C. [ 32 P]P i exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32 P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o -phthalaldehyde (OPT). Preincubation with MgADP - protected against OPT inactivation. Two potentially reactive lysine residues on the α chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding. Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein–nucleotide interactions.

The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) in the allosteric regulation of Fru-1,6-P2ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 → Ala, Glu-192 → Gln, and Asp-187 → Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to T-state transition; for wild-type Fru-1,6-P2ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered kcat from 21 s−1 for wild-type enzyme to 15 s−1 and 13 s−1, respectively, for the mutant enzymes, while their respective Km values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 → Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.

Cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase (cy-FBP/SBPase) plays a vital role in gluconeogenesis and in the photosynthetic carbon reduction pathway, and is thus a potential enzymatic target for inhibition of harmful cyanobacterial blooms. Here, we describe the crystal structure of cy-FBP/SBPase in complex with AMP and fructose-1,6-bisphosphate (FBP). The allosteric inhibitor AMP and the substrate FBP exhibit an unusual binding mode when in complex with cy-FBP/SBPase. Binding mode analysis suggested that AMP bound to the allosteric sites near the interface across the up/down subunit pairs C1C4 and C2C3 in the center of the tetramer, while FBP binds opposite to the interface between the horizontal subunit pairs C1C2 or C3C4. We identified a series of residues important for FBP and AMP binding, and suggest formation of a disulfide linkage between Cys75 and Cys99. Further analysis indicates that cy-FBP/SBPase may be regulated through ligand binding and alteration of the structure of the enzyme complex. The interactions between ligands and cy-FBP/SBPase are different from those of ligand-bound structures of other FBPase family members, and thus provide new insight into the molecular mechanisms of structure and catalysis of cy-FBP/SBPase. Our studies provide insight into the evolution of this enzyme family, and may help in the design of inhibitors aimed at preventing toxic cyanobacterial blooms.

Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223-227). The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by an approximately 9 A decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E. coli AK.

The energy sensor AMP-activated protein kinase (AMPK) is a heterotrimeric complex that is allosterically activated by AMP binding to the gamma subunit. Cocrystal structures of the mammalian AMPK core reveal occlusion of nucleotide-binding site 3 of the gamma subunit in the presence of ATP. However, site 3 is occupied in the presence of AMP. Mutagenesis studies indicate that sites 3 and 4 are important for AMPK allosteric activation.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

The catalytic conversion of ATP and AMP to ADP by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of LID and NMP domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motions of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis and multi-dimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed RMSD variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder type theoretical model to explain the observed dynamic coupling between the motions of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations., Comment: 34 pages, 9 figures

Natural products often contain unusual scaffold structures that may be elaborated by combinatorial methods to develop new drug-like molecules. Visual inspection of more than 128 natural products with some type of anti-diabetic activity suggested that a subset might provide novel scaffolds for designing potent inhibitors against fructose 1,6-bisphosphatase (FBPase), an enzyme critical in the control of gluconeogenesis. Using in silico docking methodology, these were evaluated to determine those that exhibited affinity for the AMP binding site. Achyrofuran from the South American plant Achyrocline satureoides, was selected for further investigation. Using the achyrofuran scaffold, inhibitors against FBPase were developed. Compounds 15 and 16 inhibited human liver and pig kidney FBPases at IC50 values comparable to that of AMP, the natural allosteric inhibitor.

The topological location of the nucleotide substrate binding environments on adenylate kinase has been explored with the fluorescent molecule [4-benzoyl]benzoyl-1-amidofluorescein (BzAF) and the nucleotide analog 3'-O-[4-benzoyl]benzoyl-ATP (BzATP), which are site-directed photoaffinity probes that bind covalently at the individual nucleotide sites. The MgBzATP substituted for MgATP as a substrate for this enzyme, whereas BzAF, which is neither a substrate nor a nucleotide, behaved as a competitive inhibitor for each nucleotide site independently. BzAF could be directed specifically to either of the nucleotide sites by near saturation of the second site with its natural nucleotide substrate. Using this second site blocking approach, each nucleotide site, in turn, could be protected competitively from BzAF-induced photoinhibition by the presence of its natural substrate. This strategy showed that under photolysis, the Kd(BzAF) of 0.1 mM (for the MgATP site) and 0.2 mM (for the AMP site) were nearly identical with the Km(app) values determined for MgATP and AMP, respectively. Pseudo first-order photolysis kinetics with [3H]BzAF revealed covalent binding stoichiometries for full inhibition of 1 mol of probe/mol of enzyme at either site. Thus, we prepared the binary complex ([3H]BzAF-enzyme) by photo-labeling the MgATP site-blocked enzyme with [3H]BzAf to 1:1 molar stoichiometry. Tryptic digestion followed by partial sequencing of the [3H]BzAF-labeled enzyme disclosed that BzAF was bound specifically within the peptide span Gly64-->Arg77. This amino acid domain therefore probably constitutes the neighborhood identifiable with AMP binding. Furthermore, we also achieved double photocovalent labeling of adenylate kinase with both photoprobes, by first cross-linking with approximately 1 mol of MgBzATP, followed by approximately 0.9 mol of [3H]BzAF, thus generating a ternary covalent complex. Comparison of the fluorescence of the binary species and the ternary (BzATP-enzyme-[3H]BzAF) complex revealed altered fluorescence emission profiles, which could indicate that a conformational change occurs during formation of the ternary (or transition state) complex.