Nucleotide Binding Research Articles

Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding.

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.

Activation and Autoinhibition Mechanisms of NLR Immune Receptor Pi36 in Rice.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are the most important and largest class of immune receptors in plants. The Pi36 gene encodes a canonical CC-NBS-LRR protein that confers resistance to rice blast fungal infections. Here, we show that the CC domain of Pi36 plays a role in cell death induction. Furthermore, self-association is required for the CC domain-mediated cell death, and the self-association ability is correlated with the cell death level. In addition, the NB-ARC domain may suppress the activity of the CC domain through intramolecular interaction. The mutations D440G next to the RNBS-D motif and D503V in the MHD motif autoactivated Pi36, but the mutation K212 in the P-loop motif inhibited this autoactivation, indicating that nucleotide binding of the NB-ARC domain is essential for Pi36 activation. We also found that the LRR domain is required for D503V- and D440G-mediated Pi36 autoactivation. Interestingly, several mutations in the CC domain compromised the CC domain-mediated cell death without affecting the D440G- or D503V-mediated Pi36 autoactivation. The autoactivate Pi36 variants exhibited stronger self-associations than the inactive variants. Taken together, we speculated that the CC domain of Pi36 executes cell death activities, whereas the NB-ARC domain suppressed CC-mediated cell death via intermolecular interaction. The NB-ARC domain releases its suppression of the CC domain and strengthens the self-association of Pi36 to support the CC domain, possibly through nucleotide exchange.

Constrained hidden Markov models reveal further Hsp90 protein states

Time series of conformational dynamics in proteins are usually evaluated with hidden Markov models (HMMs). This approach works well if the number of states and their connectivity is known. However, for the multi-domain protein Hsp90, a standard HMM analysis with optimization of the BIC (Bayesian information criterion) cannot explain long-lived states well. Therefore, here we employ constrained HMMs, which neglect transitions between states by including assumptions. Gradually tuning a model with justified and focused changes allows us to improve its effectiveness and the score of the BIC. This became possible by analyzing time traces with several thousand observable transitions and, therefore, superb statistics. In this scheme, we also monitor the residences in the states reconstructed by the model, aiming to find exponentially distributed dwell times. We show how introducing new states can achieve these statistics but also point out limitations, e.g. for substantial similarity of two states connected to a common neighbor. One of the states displays the lowest free energy and could be the idle open ‘waiting state’, in which Hsp90 waits for the binding of nucleotides, cochaperones, or clients.

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

Structure, dimeric conformation, and coenzyme versatility of p-hydroxybenzoate hydroxylase from Arthrobacter sp. PAMC25564

p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH's active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.

ATP/azobenzene-guanidinium self-assembly into fluorescent and multi-stimuli-responsive supramolecular aggregates

Building stimuli-responsive supramolecular systems is a way for chemists to achieve spatio-temporal control over complex systems as well as a promising strategy for applications ranging from sensing to drug-delivery. For its large spectrum of biological and biomedical implications, adenosine 5’-triphosphate (ATP) is a particularly interesting target for such a purpose but photoresponsive ATP-based systems have mainly been relying on covalent modification of ATP. Here, we show that simply mixing ATP with AzoDiGua, an azobenzene-guanidium compound with photodependent nucleotide binding affinity, results in the spontaneous self-assembly of the two non-fluorescent compounds into photoreversible, micrometer-sized and fluorescent aggregates. Obtained in water at room temperature and physiological pH, these supramolecular structures are dynamic and respond to several chemical, physical and biological stimuli. The presence of azobenzene allows a fast and photoreversible control of their assembly. ATP chelating properties to metal dications enable ion-triggered disassembly and fluorescence control with valence-selectivity. Finally, the supramolecular aggregates are disassembled by alkaline phosphatase in a few minutes at room temperature, resulting in enzymatic control of fluorescence. These results highlight the interest of using a photoswitchable nucleotide binding partner as a self-assembly brick to build highly responsive supramolecular entities involving biological targets without the need to covalently modify them.

Interaction networks within disease-associated GαS variants characterized by an integrative biophysical approach

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein–G protein-coupled receptor (GPCR) complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss-of-function or constitutive gain-of-function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed clinically-relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics (MD) simulations, which observed changes in interaction networks between GαS switch regions. Further, saturation-transfer difference NMR (STD–NMR) spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA’s association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

New Mechanisms Underlying Oncogenesis in Dbl Family Rho Guanine Nucleotide Exchange Factors.

Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis. One family of intracellular systems are the guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GTP for GDP bound to inactive guanine nucleotide binding proteins (G proteins). The resulting active G proteins then interact with downstream targets that control cell proliferation, growth, shape, migration, adhesion, and transcription. Dysregulation of any of these processes is a hallmark of cancer. The Dbl family of GEFs activates Rho family G proteins, which, in turn, alter the actin cytoskeleton and promote gene transcription. Although they have a common catalytic mechanism exercised by their highly conserved Dbl homology (DH) domains, Dbl GEFs are regulated in diverse ways, often involving the release of autoinhibition imposed by accessory domains. Among these domains, the pleckstrin homology (PH) domain is the most commonly observed and found immediately C-terminal to the DH domain. The domain has been associated with both positive and negative regulation. Recently, some atomic structures of Dbl GEFs have been determined that reemphasize the complex and central role that the PH domain can play in orchestrating regulation of the DH domain. Here, we discuss these newer structures, put them into context by cataloging the various ways that PH domains are known to contribute to signaling across the Dbl family, and discuss how the PH domain might be exploited to achieve selective inhibition of Dbl family RhoGEFs by small-molecule therapeutics. SIGNIFICANCE STATEMENT: Dysregulation via overexpression or mutation of Dbl family Rho guanine nucleotide exchange factors (GEFs) contributes to cancer and neurodegeneration. Targeting the Dbl homology catalytic domain by small-molecule therapeutics has been challenging due to its high conservation and the lack of a discrete binding pocket. By evaluating some new autoinhibitory mechanisms in the Dbl family, we demonstrate the great diversity of roles played by the regulatory domains, in particular the PH domain, and how this holds tremendous potential for the development of selective therapeutics that modulate GEF activity.

Functional genomic regions associated with blast disease resistance in rice predicted syntenic orthologs and potential resistance gene candidates from diverse cereal genomes

Blast disease is a major production constraint in cereal crops worldwide. Genome regions associated with blast resistance in rice were used in homology-based prediction of resistance gene orthologs across cereals. Seventy-four Resistance Gene Analogs (RGAs) that were collocated with rice QTLs (Quantitative Trait Loci) associated with blast resistance, predicted 89 orthologs from cereals,including 61 from finger millet. The RGA orthologs found hits in cereal transcriptomes derived from blast-infected tissues. Over 92% of the putative RGAs encoded NBS-LRR (Nucleotide Binding Site -Leucine Rich Repeat) proteins. Despite conserved primary protein structures, NBS-LRRs were variable in the encoding genes. Primers designed on conserved NBS-LRRs motifs amplified multiple fragments in PCR indicating the presence of highly similar paralogs in genomes. Rice chromosomes 1, 2, 4, 6, and 11 dock clusters of RGAs and syntenic clusters of orthologous RGAs were predicted from other cereal genomes identifying genome hotspots for resistance gene cassettes. RGAs in such cassettes were tightly linked with 50 %–90 % within-cluster similarity. Local finger millet germplasm identified multi-modes of resistance, and lack of correlation between the resistance traits points to independent expression of resistance mechanisms and associated genes in different tissues and development stages. The significant diversity, genomic abundance, and potential functional redundancy complicate use of RGAs for resistance breeding in given host genetic backgrounds, pathogen pools and agro-climates; therefore, robust technology pipelines that enable system-level analysis of expression and effectiveness can increase the practical use of RGAs.

Influence of expression and purification protocols on Gα biochemical activity: kinetics of plant and mammalian G protein cycles.

Heterotrimeric G proteins are a class of signal transduction complexes with broad roles in human health and agriculturally important plant traits. In the classic paradigm, guanine nucleotide binding to the Gα subunit regulates the activation status of the complex. Using the Arabidopsis thaliana Gα subunit, GPA1, we developed a rapid StrepII-tag mediated purification method that facilitates isolation of protein with increased enzymatic activities as compared to conventional methods, and is demonstrably also applicable to mammalian Gα subunits. We subsequently utilized domain swaps of GPA1 and human GNAO1 to demonstrate the instability of recombinant GPA1 is a function of the interaction between the Ras and helical domains, and can be partially uncoupled from the rapid nucleotide binding kinetics displayed by GPA1.

Erythrocyte membrane-camouflaged nanodelivery strategy enhances gene editing efficiency of Cas9 RNP for boosting tumor senescence

CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ). However, the inefficient precise editing of genome sequences remains a challenge for clinical treatment. Herein, an erythrocyte membrane (EM)-camouflaged and tumor microenvironment (TME)-responsive nanosystem was constructed to achieve synergistic combination of enhanced Cas9 ribonucleoprotein (RNP) gene editing efficiency and telomere dysfunction, and thus precise induced tumor inhibition. The EM-camouflaged nanosystem escaped from the reticuloendothelial system (RES) and was endocytosed by tumor cells. Azidothymidine (AZT) was gradually released as the erythrocyte shell ablation, which competitively prevented the binding of single nucleotides to the telomere replication template TR, terminate the extension of the telomerase DNA chain. Additionally, Cas9 RNP was released through disulfide bond cleavage and entered into the nucleus to realize genome editing of telomerase reverse transcriptase (TERT) gene. Simultaneous, AZT enhanced NHEJ pathway to improve the gene editing efficiency of Cas9 RNP, which further promoted the progressive telomere erosion and thus induced tumor inhibition. Overall, this biomembrane-camouflaged nanosystem realizes enhanced Cas9 RNP gene editing efficiency with high-level biosafety and promotes tumor senescence by triggering telomere disorder, which provides strategy for improving the gene editing efficiency to induce senescence of tumor cells.

Genome and transcriptome exploration reveals receptor-like kinases as potential resistance gene analogs against bacterial blight in pomegranate.

Pomegranate (Punica granatum L.) is a tropical fruit crop of pharma-nutritional importance. However, it faces farming challenges due to pests and diseases, particularly bacterial blight and wilt. Developing resistant cultivars is crucial for sustainable pomegranate cultivation, and understanding resistance's genetic basis is essential. We used an extensive resistance gene analogues (RGA) prediction tool to identify 958 RGAs, classified into Nucleotide Binding Site-leucine-rich repeat (NBS-LRR) proteins, receptor-like kinases (RLKs), receptor-like proteins (RLPs), Transmembrane coiled-coil (TM-CC), and nine non-canonical RGAs. RGAs were distributed across all eight chromosomes, with chromosome 02 containing the most RGAs (161), and chromosome 08 having the highest density (4.42 RGA/Mb). NBS-LRR genes were predominantly present on chromosomes 08 and 02, whereas RLKs and RLPs were primarily located on chromosomes 04 and 07. Gene ontology analysis revealed that 475 RGAs were associated with defence against various biotic stresses. Using RNAseq, we identified 120 differentially expressed RGAs, with RLKs (74) being prominent among the differentially expressed genes. The discovery of these RGAs is a significant step towards breeding pomegranates for pest and disease resistance. The differentially expressed RLKs hold promise for developing resistant cultivars against bacterial blight, thereby contributing to the sustainability of pomegranate cultivation.

Mechanistic insights into P-glycoprotein ligand transport and inhibition revealed by enhanced molecular dynamics simulations

P-glycoprotein (P-gp) plays a crucial role in cellular detoxification and drug efflux processes, transitioning between inward-facing (IF) open, occluded, and outward-facing (OF) states to facilitate substrate transport. Its role is critical in cancer therapy, where P-gp contributes to the multidrug resistance phenotype. In our study, classical and enhanced molecular dynamics (MD) simulations were conducted to dissect the structural and functional features of the P-gp conformational states. Our advanced MD simulations, including kinetically excited targeted MD (ketMD) and adiabatic biasing MD (ABMD), provided deeper insights into state transition and translocation mechanisms. Our findings suggest that the unkinking of TM4 and TM10 helices is a prerequisite for correctly achieving the outward conformation. Simulations of the IF-occluded conformations, characterized by kinked TM4 and TM10 helices, consistently demonstrated altered communication between the transmembrane domains (TMDs) and nucleotide binding domain 2 (NBD2), suggesting the implication of this interface in inhibiting P-gp's efflux function. A particular emphasis was placed on the unstructured linker segment connecting the NBD1 to TMD2 and its role in the transporter's dynamics. With the linker present, we specifically noticed a potential entrance of cholesterol (CHOL) through the TM4-TM6 portal, shedding light on crucial residues involved in accommodating CHOL. We therefore suggest that this entry mechanism could be employed for some P-gp substrates or inhibitors. Our results provide critical data for understanding P-gp functioning and developing new P-gp inhibitors for establishing more effective strategies against multidrug resistance.

The role of microRNAs in NBS-LRR gene expression and its implications for plant immunity and crop development.

Plants evolved, over millions of years, complex defense systems against pathogens. Once infected, the interaction between pathogen effector molecules and host receptors triggers plant immune responses, which include apoptosis, systemic immune response, among others. An important protein family responsible for pathogen effector recognition is the nucleotide binding site-leucine repeat rich (NBS-LRR) proteins. The NBS-LRR gene family is the largest disease resistance gene class in plants. These proteins are widely distributed in vascular plants and have a complex multigenic cluster distribution in plant genomes. To counteract the genetic load of such a large gene family on fitness cost, plants evolved a mechanism using post transcriptional gene silencing induced by small RNAs, particularly microRNAs. For the NBS-LRR gene family, the small RNAs involved in this silencing mechanism are mainly the microRNA482/2118 superfamily. This suppression mechanism is relieved upon pathogen infection, thus allowing increased NBS-LRR expression and triggering plant immunity. In this review, we will discuss the biogenesis of microRNAs and secondary RNAs involved in this silencing mechanism, biochemical and structural features of NBS-LRR proteins in response to pathogen effectors and the evolution of microRNA-based silencing mechanism with a focus on the miR482/2118 family. Furthermore, the biotechnological manipulation of microRNA expression, using both transgenic or genome editing approaches to improve cultivated plants will be discussed, with a focus on the miR482/2118 family in soybean.

An atomistic model of myosin interacting heads motif dynamics and their modification by 2'-deoxy-ADP.

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are unable to perform motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state via missense mutations can pathologically disrupt the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2'-deoxy-ATP (dATP) is a potent myosin activator which destabilizes the IHM. Here we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations with IHM containing ADP.Pi in both nucleotide binding pockets revealed residual dynamics in an otherwise 'inactive' and 'sequestered' state of a motor protein. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that modify the protein-protein interface that stabilizes the sequestered state, and changes to this interface were accompanied by allosteric changes in remote regions of the protein complex. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.

Correction to “Guanine nucleotide–binding protein G(i)a2 aggravates hepatic ischemia‐reperfusion injury in mice by regulating MLK3 signaling”

Correction to “Guanine nucleotide–binding protein G(i)a2 aggravates hepatic ischemia‐reperfusion injury in mice by regulating <scp>MLK3</scp> signaling”

Growth factor-dependent phosphorylation of Gαi shapes canonical signaling by G protein-coupled receptors.

A long-standing question in the field of signal transduction is how distinct signaling pathways interact with each other to control cell behavior. Growth factor receptors and G protein-coupled receptors (GPCRs) are the two major signaling hubs in eukaryotes. Given that the mechanisms by which they signal independently have been extensively characterized, we investigated how they may cross-talk with each other. Using linear ion trap mass spectrometry and cell-based biophysical, biochemical, and phenotypic assays, we found at least three distinct ways in which epidermal growth factor affected canonical G protein signaling by the Gi-coupled GPCR CXCR4 through the phosphorylation of Gαi. Phosphomimicking mutations in two residues in the αE helix of Gαi (tyrosine-154/tyrosine-155) suppressed agonist-induced Gαi activation while promoting constitutive Gβγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed Gi activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gαi proteins, including conformational stability, nucleotide binding, and the ability to associate with and to release Gβγ. However, one phosphomimicking mutation, targeting the carboxyl-terminal residue tyrosine-320, promoted mislocalization of Gαi from the plasma membrane, a previously uncharacterized mechanism of suppressing GPCR signaling through G protein subcellular compartmentalization. Together, these findings elucidate not only how growth factor and chemokine signals cross-talk through the phosphorylation-dependent modulation of Gαi but also how such cross-talk may generate signal diversity.

A unique mode of coenzyme A binding to the nucleotide binding pocket of human metastasis suppressor NME1

A unique mode of coenzyme A binding to the nucleotide binding pocket of human metastasis suppressor NME1