Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase.

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

Multiscale modeling shows how 2'-deoxy-ATP rescues ventricular function in heart failure.

2'-deoxy-ATP (dATP) improves cardiac function by increasing the rate of crossbridge cycling and Ca[Formula: see text] transient decay. However, the mechanisms of these effects and how therapeutic responses to dATP are achieved when dATP is only a small fraction of the total ATP pool remain poorly understood. Here, we used a multiscale computational modeling approach to analyze the mechanisms by which dATP improves ventricular function. We integrated atomistic simulations of prepowerstroke myosin and actomyosin association, filament-scale Markov state modeling of sarcomere mechanics, cell-scale analysis of myocyte Ca[Formula: see text] dynamics and contraction, organ-scale modeling of biventricular mechanoenergetics, and systems level modeling of circulatory dynamics. Molecular and Brownian dynamics simulations showed that dATP increases the actomyosin association rate by 1.9 fold. Markov state models predicted that dATP increases the pool of myosin heads available for crossbridge cycling, increasing steady-state force development at low dATP fractions by 1.3 fold due to mechanosensing and nearest-neighbor cooperativity. This was found to be the dominant mechanism by which small amounts of dATP can improve contractile function at myofilament to organ scales. Together with faster myocyte Ca[Formula: see text] handling, this led to improved ventricular contractility, especially in a failing heart model in which dATP increased ejection fraction by 16% and the energy efficiency of cardiac contraction by 1%. This work represents a complete multiscale model analysis of a small molecule myosin modulator from single molecule to organ system biophysics and elucidates how the molecular mechanisms of dATP may improve cardiovascular function in heart failure with reduced ejection fraction.

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.

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

Modulation of post-powerstroke dynamics in myosin II by 2'-deoxy-ADP.

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle.

Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9.

Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs.

Telomerase is a specialized reverse transcriptase that adds GGTTAG repeats to chromosome ends and is upregulated in most human cancers to enable limitless proliferation. Here, we uncover two distinct mechanisms by which naturally occurring oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation. We conduct a series of direct telomerase extension assays in the presence of modified dNTPs on various telomeric substrates. We provide direct evidence that telomerase can add the nucleotide reverse transcriptase inhibitors ddITP and AZT-TP to the telomeric end, causing chain termination. In contrast, telomerase continues elongation after inserting oxidized 2-OH-dATP or therapeutic 6-thio-dGTP, but insertion disrupts translocation and inhibits further repeat addition. Kinetics reveal that telomerase poorly selects against 6-thio-dGTP, inserting with similar catalytic efficiency as dGTP. Furthermore, telomerase processivity factor POT1-TPP1 fails to restore processive elongation in the presence of inhibitory dNTPs. These findings reveal mechanisms for targeting telomerase with modified dNTPs in cancer therapy.

MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool.

MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.

Mycobacterium tuberculosis thymidylate synthase (ThyX) is a target for plumbagin, a natural product with antimycobacterial activity.

Plumbagin derived from the plant Plumbago indica, known as Chitrak in India, is an example of a medicinal compound used traditionally to cure a variety of ailments. Previous reports have indicated that it can inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent of the deadly disease TB. In this investigation, we provide an insight into its mode of action. We show here that a significant mycobacterial target that is inhibited by plumbagin is the enzyme ThyX, a form of thymidylate synthase, that is responsible for the synthesis of dTMP from dUMP in various bacterial pathogens, including Mtb. Using a purified preparation of the recombinant version of Mtb ThyX, we demonstrate that plumbagin, a 2,4 napthoquinone, but not lawsone, a structurally related medicinal compound, inhibits its activity in vitro. We also show that the intracellular [dTTP]/[dATP] ratio in Mycobacterium smegmatis (Msm) cells decrease upon treatment with plumbagin, and this, in turn, leads to cell death. Such a conclusion is supported by the observation that over-expression of thyx in the plumbagin treated Msm cells leads to the restoration of viability. The results of our investigation indicate that plumbagin kills mycobacterial cells primarily by targeting ThyX, a vital enzyme required for their survival.

Mechanism of Activation of AMPK by Cordycepin.

Cordycepin (3'-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.

Mutual role of ecto-5'-nucleotidase/CD73 and concentrative nucleoside transporter 3 in the intestinal uptake of dAMP.

2'-Deoxyadenosine 5'-monophosphate (dAMP), a deoxyribonucleotide found in DNA, affects intestinal cell growth. The molecular mechanisms underlying gastrointestinal absorption of foreign DNA ingested along with food has hardly been investigated. The aim of this study was to investigate the mechanism underlying intestinal absorption of dAMP. The uptake of [3H]dAMP by Caco-2 cells was Na+- and pH-dependent and was inhibited by various nucleosides. In contrast, nitrobenzylthioinosine (NMBPR), an equilibrative nucleoside transporter inhibitor, showed little inhibitory effects on [3H]dAMP uptake. Additionally, human concentrative nucleoside transporter (CNT) 3, transiently expressed in COS-7 cells, mediated the uptake of [3H]dAMP. A kinetic study revealed that the Km value of CNT3-mediated uptake of dAMP (59.6 µM) was close to that of 2'-deoxyadenosine (dAdo) (56.3 µM), whereas the dAMP Vmax (15.6 pmol·mg protein-1min-1) was 500-fold lesser than the dAdo Vmax (7782 pmol·mg protein-1min-1). Further, [3H]dAMP uptake was greater in COS-7 cells expressing ecto-5'-nucleotidase/CD73 with CNT3 than in those expressing CNT3 alone. These data suggest that, although dAMP is a substrate of CNT3, it is dephosphorylated to dAdo by CD73 and is efficiently absorbed as dAdo from the intestinal lumen.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

A Transition-State Perspective on Y-Family DNA Polymerase η Fidelity in Comparison with X-Family DNA Polymerases λ and ß.

Deoxynucleotide misincorporation efficiencies can span a wide 104-fold range, from ∼10-2 to ∼10-6, depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our "tool-kit" of dATP- and dGTP-ß,γ substrate analogues in which the pyrophosphate leaving group (p Ka4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p Ka4 values from 7.8 (CF2) to 12.3 [C(CH3)2]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( kpol) versus p Ka4, for Y-family error-prone pol η and X-family pols λ and ß to determine the extent to which different electrostatic active site environments alter kpol values. The apparent chemical rate constant ( kpol) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show kpol values that are systematically lower than would be predicted from their p Ka4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p Ka4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.

The invariant glutamate of human PrimPol DxE motif is critical for its Mn2+-dependent distinctive activities.

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg2+ or Mn2+, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp114, Glu116 and Asp280) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu116 as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn2+. Herein, we evidenced that PrimPol Glu116 contributes to error-prone tolerance of 8oxodG more markedly when both Mg2+ and Mn2+ ions are present. Moreover, Glu116 was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn2+ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu116 was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn2+ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.

ELTA: Enzymatic Labeling of Terminal ADP-Ribose.

ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2'-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates.

A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant.

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor MnIII/MnIV We show that NrdA from F. ignava can form a catalytically competent RNR with the MnIII/MnIV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.

An endogenous dAMP ligand in Bacillus subtilis class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP.

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme's quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5'-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α2 dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner.

Second-Shell Basic Residues Expand the Two-Metal-Ion Architecture of DNA and RNA Processing Enzymes.

Synthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution. We discuss possible mechanistic implications of this expanded two-metal-ion architecture, including inferences on medium-resolution cryoelectron microscopy structures. Ultimately, our analysis may inspire future experiments and strategies for enzyme engineering or drug design to modulate nucleic acid processing.

Gp41, a superfamily SF2 helicase from bacteriophage BFK20.

Gp41 is one of two helicases encoded by the genome of bacteriophage BFK20. The gp41 sequence contains conserved motifs from the SF2 family of helicases. We prepared and studied three recombinant proteins: gp41HN, a wild type-like protein with an N-terminal His-Tag; gp41HC, with an S2A mutation and a C-terminal His-Tag; and gp41dC, a mutant protein with a deleted C-terminal region and His-Tags on both N- and C-termini. We tested the enzymatic activities and DNA binding abilities of these isolated proteins. We found that both gp41HN and gp41HC had strong DNA-dependent ATPase activities, but that the ATPase activity of gp41dC was significantly lower regardless of the presence of DNA. The preferred substrates for the NTP hydrolysis reactions were ATP and dATP. gp41HC and gp41HN exhibited a low helicase activity in a fluorescence-based assay using dsDNA substrates with a 3' overhang and with a forked end in the presence of ATP. We infer that the C-terminal region of gp41 may be involved in DNA binding, since removing this region in gp41dC reduced the protein's DNA binding ability.

Identification of a Different Agonist-Binding Site and Activation Mechanism of the Human P2Y1 Receptor.

The human P2Y1 receptor (P2Y1R) is a purinergic G-protein-coupled receptor (GPCR) that functions as a receptor for adenosine 5'-diphosphate (ADP). An antagonist of P2Y1R might potentially have antithrombotic effects, whereas agonists might serve as antidiabetic agents. On the basis of the antagonist-bound MRS2500-P2Y1R crystal structure, we constructed computational models of apo-P2Y1R and the agonist-receptor complex 2MeSADP-P2Y1R. We then performed conventional molecular dynamics (cMD) and accelerated molecular dynamics (aMD) simulations to study the conformational dynamics after binding with agonist/antagonist as well as the P2Y1R activation mechanism. We identified a new agonist-binding site of P2Y1R that is consistent with previous mutagenesis data. This new site is deeper than those of the agonist ADP in the recently simulated ADP-P2Y1R structure and the antagonist MRS2500 in the MRS2500-P2Y1R crystal structure. During P2Y1R activation, the cytoplasmic end of helix VI shifts outward 9.1 Å, the Ser1463.47-Tyr2375.58 hydrogen bond breaks, a Tyr2375.58-Val2626.37 hydrogen bond forms, and the conformation of the χ1 rotamer of Phe2696.44 changes from parallel to perpendicular to helix VI. The apo-P2Y1R system and the MRS2500-P2Y1R system remain inactive. The newly identified agonist binding site and activation mechanism revealed in this study may aid in the design of P2Y1R antagonists/agonists as antithrombotic/antidiabetic agents, respectively.