AMP Binding Research Articles

A Proteomic Study on Seed Germination of Nitraria roborowskii Kom.

Owing to the dormancy of the seeds of Nitraria roborowskii Kom., which results in a low germination rate in nature, germination takes a long time, and natural regeneration is difficult. Therefore, there is a need to study the molecular mechanism by which the seeds of N. roborowskii release dormancy. In this study, the differentially expressed proteins of N. roborowskii seeds before and after dormancy and germination were quantitatively and qualitatively analyzed via two-dimensional high-performance liquid chromatography tandem mass spectrometry (LC–MS) and TMTTM. Differentially expressed proteins from dormant and germinated seeds were characterized and enriched via bioinformatics to determine the functions and pathways of the differentially expressed proteins. The results revealed that seed dormancy was regulated by multiple metabolic pathways, including protein synthesis, nutrient utilization and phytohormone signal transduction pathways. A comparison of the dormant and germinated N. roborowskii seed samples revealed 1082 differentially expressed proteins with FC ≥ 1.5 and p values ≤ 0.05, among which 191 proteins were upregulated and 891 proteins were downregulated in the seeds of the germinated group, and proteins more closely related to the key genes of the germinated N. roborowskii seeds were involved in the activity of D-threo-aldose 1-dehydrogenase. Four proteins (WD40, cystatin, AMP binding protein, and helicase) were involved in the positive regulation of seed germination. The release of N. roborowskii seed dormancy is a complex biological process involving cell differentiation, formation, cellular transport, signaling and resistance, etc. The interactions of multiple metabolic pathways, such as carbon fixation, glycolysis/gluconeogenesis, the pentose phosphate pathway, endoplasmic reticulum protein processing and pyruvic acid metabolism in photosynthetic organisms, constitute a complex regulatory mechanism for dormancy release.

Structural insights into BirA from Haemophilus influenzae, a bifunctional protein as a biotin protein ligase and a transcriptional repressor

Biotin is an essential coenzyme involved in various metabolic processes across all known organisms, with biotinylation being crucial for the activity of carboxylases. BirA from Haemophilus influenzae is a bifunctional protein that acts as a biotin protein ligase and a transcriptional repressor. This study reveals the crystal structures of Hin BirA in both its apo- and holo-(biotinyl-5′-AMP bound) forms. As a class II BirA, it consists of three domains: N-terminal DNA binding domain, central catalytic domain, and C-terminal SH3-like domain. The structural analysis shows that the biotin-binding loop forms an ordered structure upon biotinyl-5′-AMP binding. This facilitates its interaction with the ligand and promotes protein dimerization. Comparative studies with other BirA homologs from different organisms indicate that the residues responsible for binding biotinyl-5′-AMP are highly conserved. This study also utilized AlphaFold2 to model the potential heterodimeric interaction between Hin BirA and biotin carboxyl carrier protein, thereby providing insights into the structural basis for biotinylation. These findings enhance our understanding of the structural and functional characteristics of Hin BirA, highlighting its potential as a target for novel antibiotics that disrupt the bacterial biotin synthesis pathways.

AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

Cyclic AMP binding to a universal stress protein in Mycobacterium tuberculosis is essential for viability

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis andMycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M.tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M.smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M.smegmatis, we found that the rv1636 gene is essential for the viability of M.tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.

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

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

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei.

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.

SLC27 family of fatty acid transporters in GtoPdb v.2023.1

Fatty acid transporter proteins (FATPs) are a family (SLC27) of six transporters (FATP1-6). They have at least one, and possibly six [6, 13], 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) [4, 10, 11]. 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 [9, 13, 1].

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Comparison of cyclic AMP binding activity within HCN channel subfamily using atomistic molecular dynamics simulations of isolated cyclic nucleotide binding domains.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a subfamily of homotetrameric, voltage gated potassium/sodium channels whose activity is modulated by cyclic AMP (cAMP). The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region which is induced by the binding of cAMP is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that affects conductance. Although various static structures of the four HCN isoforms with and without bound cyclic nucleotide have been solved and deposited on the Protein Data Bank, there is still a need to understand the precise binding interactions which lead to different sensitivities and selectivities for cyclic nucleotides within the subfamily. Using atomistic molecular dynamics, we have simulated the isolated CNBD of HCN1-4 bound to cAMP and have used both equilibrium simulations and alchemical free energy perturbation to characterize and compare the binding activities of the different isoforms. These simulations highlight the differential cyclic nucleotide binding behavior between HCN channels.

Identification of Key Modules and Candidate Genes for Powdery Mildew Resistance of Wheat-Agropyron cristatum Translocation Line WAT-2020-17-6 by WGCNA

As one of the serious diseases of wheat, powdery mildew (Blumeria graminis f. sp. tritici) is a long-term threat to wheat production. Therefore, it is of great significance to explore new powdery mildew-resistant genes for breeding. The wild relative species of wheat provide gene resources for resistance to powdery mildew breeding. Agropyron cristatum (2n = 4x = 28, genomes PPPP) is an important wild relative of wheat, carrying excellent genes for high yield, disease resistance, and stress resistance, which can be used for wheat improvement. To understand the molecular mechanism of powdery mildew resistance in the wheat-A. cristatum translocation line WAT2020-17-6, transcriptome sequencing was performed, and the resistance genes were analyzed by weighted gene co-expression network analysis (WGCNA). In the results, 42,845 differentially expressed genes were identified and divided into 18 modules, of which six modules were highly correlated with powdery mildew resistance. Gene ontology (GO) enrichment analysis showed that the six interested modules related to powdery mildew resistance were significantly enriched in N-methyltransferase activity, autophagy, mRNA splicing via spliceosome, chloroplast envelope, and AMP binding. The candidate hub genes of the interested modules were further identified, and their regulatory relationships were analyzed based on co-expression data. The temporal expression pattern of the 12 hub genes was verified within 96 h after powdery mildew inoculation by RT-PCR assay. In this study, we preliminarily explained the resistance mechanism of the wheat-A. cristatum translocation lines and obtained the hub candidate genes, which laid a foundation in the exploration of resistance genes in A. cristatum for powdery mildew-resistant breeding in wheat.

Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP‐glucose pyrophosphorylase. By X‐ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate‐binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP‐glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.

Impact of Redox Modification on PKA Substrate Selection

Protein kinases regulate multiple signaling pathways involved in health and disease. For instance, cyclic adenosine monophosphate‐dependent protein kinase (PKA) controls a diverse set of cellular functions, including gene expression, cell cycle progression, metabolism, and apoptosis. Dysregulation of PKA has been linked to many diseases, including diabetes, cancer, and neurodegenerative disorders. PKA is a tetramer consisting of two regulatory and two catalytic subunits. Cyclic AMP binding to the regulatory subunits leads to dissociation of the catalytic subunits, unleashing them to phosphorylate serine and threonine residues on their target substrates. Previous studies suggest that the alpha isoform of the PKA catalytic subunit (PKA Cα) is oxidized on C199 within the P+1 substrate binding region. We hypothesized that, by modifying the substrate selectivity of PKA Cα, oxidation may regulate which pathway is activated in response to a given signal. Specifically, we investigated the impact of redox modification on the substrate selection of PKA Cα. To this end, we used fluorescence polarization (FP) spectroscopy to assess the ability of oxidized and reduced forms of PKA Cα to bind a variety of model peptide ligands (e.g., PKI, Kemptide, and CREBtide). The binding of each ligand was monitored under equilibrium binding conditions and the apparent equilibrium binding parameters (i.e., KD,app) were determined for each kinase‐ligand pair. These studies suggest that H2O2‐dependent oxidation of PKA Cα alters its interactions with some ligands while having little effect on others. To gain further insights into the factors driving the differential changes in the interaction between PKA Cα and its ligands, we are currently using surface plasmon resonance (SPR) to measure changes in the apparent rate constants, kon,app and koff,app, as well as KD,app. Together, these studies offer important insights into the mechanisms of crosstalk between redox and phosphorylation‐dependent signaling at the level of kinase substrate selection and have implications for kinase regulation during normal and pathological states. Interestingly, the site of oxidation is highly conserved among AGC kinase family members. Therefore, in the future, we will use a multipronged approach to explore the impact of redox modification on the substrate selection of other AGC family members, such as PKA Cβ and AKT1, in neuronal signaling and the etiology of various diseases.

Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the “electron transfer” (ET) and the “bifurcating” (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop (“zipper”) and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.

ALG2 regulates type I interferon responses by inhibiting STING trafficking.

Stimulator of IFN genes (STING), an endoplasmic reticulum (ER) signaling adaptor, is essential for the type I interferon response to cytosolic double-stranded DNA. Translocation from the ER to perinuclear vesicles following cyclic GMP-AMP (cGAMP) binding is a critical step for STING to activate downstream signaling molecules, which leads to the production of interferon and pro-inflammatory cytokines. Here, we found that apoptosis-linked gene 2 (ALG2, also known as PDCD6) suppressed STING signaling induced by herpes simplex virus-1 (HSV-1) infection or cGAMP presence. Knockout of ALG2 markedly increased the expression of type I interferons upon cGAMP treatment or HSV-1 infection in THP-1 monocytes. Mechanistically, ALG2 associated with the C-terminal tail of STING and inhibited its trafficking from the ER to the perinuclear region. Furthermore, the ability of ALG2 to coordinate Ca2+ was crucial for its regulation of STING trafficking and DNA-induced innate immune responses. This work suggests that ALG2 is involved in DNA-induced innate immune responses by regulating STING trafficking.

Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK

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.

Identification and expression analysis of pathogenicity-related genes of Rhizoctonia solani anastomosis groups infecting rice.

The online version contains supplementary material available at 10.1007/s13205-021-02934-1.

Abstract LB174: Discovery and preclinical characterization of INCA00186, a humanized monoclonal antibody antagonist of CD73, as a cancer immunotherapy

Abstract In the tumor microenvironment, dying cells release ATP which gets converted to AMP by CD39. AMP is subsequently converted to immunosuppressive adenosine by the extracellular 5'-nucleotidase CD73. CD73 is linked to the cell surface by a GPI-anchor and can be released into the plasma as soluble CD73. In order to lower extracellular levels and reverse the immunosuppressive activity of adenosine in the tumor microenvironment, an antibody was researched and discovered that antagonizes CD73 function. INCA00186 is a humanized monoclonal antibody that binds and inhibits CD73 function. INCA00186 binds human and cynomolgus CD73 with sub-nanomolar affinity, and inhibits CD73 enzymatic activity in a manner that is non-competitive for AMP binding. In functional studies, INCA00186 restored effector T cell proliferation in the presence of high concentrations of AMP. In the human A375 melanoma tumor model in CD34+ humanized NSG mice, INCA00186 decreased levels of cell surface CD73, displayed high receptor occupancy on tumor cells, and decreased CD73 activity in ex vivo assays of tumor homogenates. In the human MDA-MB-231 breast tumor model in CD34+ humanized NSG mice, INCA00186 decreased intratumoral adenosine concentrations. In combination with the novel A2A/A2B adenosine receptor antagonist INCB106385, INCA00186 synergistically restored effector T cell activity as measured by interferon gamma (IFNγ) production in the presence of high concentrations of AMP. In the human MDA-MB-231 breast tumor model in CD34+ humanized NSG mice, combination treatment with INCA00186 and INCB106385 controlled tumor growth significantly better than monotherapies. In summary, the data presented in this study demonstrates that INCA00186 is a potent CD73 antagonist and effectively attenuates adenosine induced immunosuppression in the tumor microenvironment. INCA00186 demonstrated anti-tumor immunity alone and, to a greater extent, in combination with the A2A/A2B adenosine receptor antagonist INCB106385. Citation Format: Shaun Stewart, Rebecca Buonpane, Jing Zhou, Michael Hansbury, Michael Smith, Hui Wang, Lu, Bin Su, Rahel Awdew, Cheng-Yen Huang, Ashwini Kulkarni, Shane Harvey, Arpita Mondal, Steve Wang, Christina Stevens, Michael Pratta, Elham Behshad, Alexandra Fanuka, Xiaodi Ren, Holly Koblish, Horacio Nastri, Patrick Mayes. Discovery and preclinical characterization of INCA00186, a humanized monoclonal antibody antagonist of CD73, as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB174.

Investigation of Cyclic AMP Binding Interactions with Isolated Cyclic Nucleotide Binding Domain of HCN1 Channel using Atomistic Molecular Dynamics Simulations at Microsecond Timescale

Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) is a homotetrameric, voltage-gated potassium/sodium channel whose activity is modulated by cyclic AMP. The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region, which is induced by the binding of cAMP, is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that leads to increased conductance. Although static structures of both the apo and holo channel states have been solved and compared, there is still a need to understand the precise binding interactions which occur between the cyclic nucleotide and the binding pocket, as well as the dynamic changes to the local protein structure that might lead to the aforementioned changes in activity. Using atomistic molecular dynamics simulations, we have simulated both the apo and holo forms of the isolated CNBD region at a microsecond timescale, and have performed a variety of analyses to relate the two conformations and investigate the interactions of the ligand with the binding pocket. We have also used enhanced sampling techniques to calculate the binding free energy of cAMP. This study provides a computational framework for the study of HCN1-ligand interactions.

Structural insights into a new substrate binding mode of a histidine acid phosphatase from Legionella pneumophila

MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapAD281A mutant in complex with 5′-AMP were solved at 2.2 and 2.6 Å resolution, respectively. The structure of the MapAD281A/5′-AMP complex reveals that the 5′-AMP fits fully into the substrate binding tunnel, with the 2′-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5′-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs.

Structure based identification of first-in-class fragment inhibitors that target the NMN pocket of M. tuberculosis NAD+-dependent DNA ligase A

NAD+-dependent DNA ligase (LigA) is the essential replicative ligase in bacteria and differs from ATP-dependent counterparts like the human DNA ligase I (HligI) in several aspects. LigA uses NAD+ as the co-factor while the latter uses ATP. Further, the LigA carries out enzymatic activity with a single divalent metal ion in the active site while ATP-dependent ligases use two metal ions. Instead of the second metal ion, LigA have a unique NMN binding subdomain that facilitates the orientation of the β-phosphate and NMN leaving group. LigA are therefore attractive targets for new anti-bacterial therapeutic development. Others and our group have earlier identified several LigA inhibitors that mainly bind to AMP binding site of LigA. However, no inhibitor is known to bind to the unique NMN binding subdomain. We initiated a fragment inhibitor discovery campaign against the M. tuberculosis LigA based on our co-crystal structure of adenylation domain with AMP and NMN. The study identified two fragments, 4-(4-fluorophenyl)-4,5,6,7-tetrahydro-3H imidazo[4,5-c] pyridine and N-(4-methylbenzyl)-1H-pyrrole-2-carboxamide, that bind to the NMN site. The fragments inhibit LigA with IC50 of 16.9 and 28.7 µM respectively and exhibit MIC of ~20 and 60 µg/ml against a temperature sensitive E. coli GR501 ligAts strain, rescued by MtbLigA. Co-crystal structures of the fragments with the adenylation domain of LigA show that they mimic the interactions of NMN. Overall, our results suggest that the NMN binding-site is a druggable target site for developing anti-LigA therapeutic strategies.