Adenine Nucleotide Binding Research Articles

Homologous Binding Sites in Yeast Isocitrate Dehydrogenase for Cofactor (NAD+) and Allosteric Activator (AMP)

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

Molecular Architecture of the Phosphorylation Region of the Yeast Plasma Membrane H+-ATPase

The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage. An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2). Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+). The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively. The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP. Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes.

Cooperative nucleotide binding to the human erythrocyte sugar transporter.

The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter.

The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover

As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999) J. Biol. Chem. 274, 12209-12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001) J. Biol. Chem. 276, 12918-12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5'-adenyl-beta,gamma-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg(2+)-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN(3)ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N(3)AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis.

How does the mitochondrial ADP/ATP carrier distinguish transportable ATP and ADP from untransportable AMP and GTP?Dynamic modeling of the recognition/translocation process in the major substrate binding region

To understand the transport mechanism of the bovine heart mitochondrial ADP/ATP carrier at the atomic level, we studied the four-dimensional features of the interaction of various purine nucleotides with the adenine nucleotide binding region (ABR) consisting of Arg 151-Asp 167 in the second loop facing the matrix side. After three-dimensional modeling of ABR based on the experimental results, its structural changes on interaction with purine nucleotides were examined by molecular dynamics computation at 300 K. ATP/ADP were translocated to a considerable degree from the matrix side to the inner membrane region accompanied by significant backbone conformational changes, whereas neither appreciable translocation nor a significant conformational change was observed with the untransportable nucleotides AMP/GTP. The results suggested that binding of the terminal phosphate group and the adenine ring of ATP/ADP with Arg 151 and Lys 162, respectively, and subsequent interaction of a phosphate group(s) other than the terminal phosphate with Lys 162 triggered the expansion and subsequent contraction of the backbone conformation of ABR, leading to the translocation of ATP/ADP. Based on a simplified molecular dynamic simulation, we propose a dynamic model for the initial recognition process of ATP/ADP with the carrier.

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface.

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

The permeability transition pore complex: another view

Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix 〚Ca 2+〛, especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl–prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca 2+ or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys 56 and Cys 159) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys 159 modification whilst oxidation of Cys 56 increases CyP-D binding to the ANT, probably at Pro 61.

A site-directed mutagenesis analysis of tNOX functional domains

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX ( t umor-associated N ADH ox idase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

Identification of the ABC protein SapD as the subunit that confers ATP dependence to the K+-uptake systems Trk(H) and Trk(G) from Escherichia coli K-12.

The activity of the two almost identical K+-uptake systems, Trk(H) and Trk(G), from Escherichia coli K-12 depends completely and partially on the presence of the trkE gene, respectively. trkE maps inside the sapABCDF operon, which encodes an ATP-binding cassette (ABC) transporter of unknown function from the subgroup of peptide-uptake systems. This study was carried out to clarify the role of sapABCDF gene products in the ATP dependence of the E. coli Trk systems. For this purpose DeltasapABCDF DeltatrkG and DeltasapABCDF DeltatrkH strains of E. coli containing plasmids with sap genes from either E. coli or Vibrio alginolyticus were used. All five plasmid-encoded E. coli Sap proteins were made in E. coli mini-cells. The presence of the ATP-binding SapD protein from either E. coli or V. alginolyticus alone was sufficient for stimulating the K+ transport activity of the Trk(H) and Trk(G) systems. K+-uptake experiments with Escherichia coli cells containing SapD variants with changes in the Walker A box Lys-46 residue, the Walker B box Asp-183 residue and the signature motif residues Gly-162 or Gln-165 suggested that adenine nucleotide binding to SapD rather than ATP hydrolysis by this subunit is required for the activity of the E. coli Trk(H) system. K+ transport via two plasmid-encoded Trk systems in a DeltasapABCDF E. coli strain remained dependent on both a high membrane potential and a high cytoplasmic ATP concentration, indicating that in E. coli ATP dependence of Trk activity can be independent of Sap proteins. These data are interpreted to mean that Trk systems can interact with an ABC protein other than SapD.

Point Mutations in a Hinge Linking the Small and Large Domains of β-Actin Result in Trapped Folding Intermediates Bound to Cytosolic Chaperonin CCT

The30-Å cryo-EM-derived structure of apo-CCT–α-actin shows actin opened up across its nucleotide-binding cleft and binding to either of two CCT subunit pairs, CCTβ–CCTδ or CCTϵ–CCTδ, in a similar 1:4 arrangement. The two main duplicated domains of native actin are linked twice, topologically, by the connecting residues, Q137–S145 and P333–S338, and are tightly held together by hydrogen bonding with bound adenine nucleotide. We carried out a mutational screen to find residues in actin that might be involved in the huge rotations observed in the CCT-bound folding intermediate. When two evolutionarily highly conserved glycine residues of β-actin, G146 and G150, were changed to proline, both mutant actin proteins were poorly processed by CCT in in vitro translation assays; they become arrested on CCT. A three-dimensional reconstruction of the substrate-bound ring of the apo-CCT–β-actin complex shows that β-actin G150P is not able to bind across the chaperonin cavity to interact with the CCTδ subunit. β-actin G150P seems tightly packed and apparently bound only to the CCTβ and CCTϵ subunits, which further indicates that these CCT subunits drive the interaction between CCT and actin. Hinge opening seems to be critical for actin folding, and we suggest that residues G146 and G150 are important components of the hinge around which the rigid subdomains, presumably already present in early actin folding intermediates, rotate during CCT-assisted folding.

Early life of cystic fibrosis transmembrane conductance regulator (CFTR) in the cell

Growing up to be a mature and stable ion channel appears to be especially difficult for the cystic fibrosis transmembrane conductance regulator (CFTR)1.Riordan J.R. Cystic fibrosis as a disease of misprocessing of the cystic fibrosis transmembrane conductance regulator glycoprotein.Am J Hum Genet. 1999; 64: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar. Whereas other adenine nucleotide binding cassette (ABC) transporters including members of the same subfamily (ABCC) mature efficiently, only about 30% of wild-type nascent CFTR polypeptides mature and proceed beyond the endoplasmic reticulum (ER) in mammalian cells. The remaining 70% are polyubiquitinated and degraded by the proteasome2.Jensen T.J. Loo M.A. Pind S. et al.Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (742) Google Scholar. The δF508 present in 90% of cystic fibrosis (CF) patients and many different missense mutations completely preclude maturation. Although detectable at steady state, the immature core-glycosylated form of neither the wild-type nor δF508 CFTR accumulate in the ER. There is some evidence from both mammalian cells and yeast that nascent CFTR may be “retrotranslocated” from the ER membrane enabling proteolysis by ER-associated proteasomes. Crucial questions that need to be answered to understand the biosynthetic lability of CFTR include (1) what are the features of the molecule that prevent it from maturing efficiently, and (2) how are molecules that can mature distinguished from those that cannot? CFTR most obviously differs from other ABCC family members by the presence of charged amino acids in membrane-spanning sequences, the large central R-domain rich in phosphorylation sites and an extended C-terminal domain beyond the second nucleotide-binding domain. Phosphorylation sites of the R-domain apparently play no crucial role in maturation3.Seibert F.S. Chang X.-B. Aleksandrov A.A. et al.Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum.Biochim Biophys Acta. 1999; 1461: 275-283Crossref PubMed Scopus (62) Google Scholar. C-terminal sequences were shown to impact maturation; however, a truncated CFTR molecule without C-terminal domain shows still a substantial degree of maturation4.Gentzsch M. Riordan J.R. Localization of sequences within the C-terminal domain of CFTR which impact maturation and stability.J Biol Chem. 2001; 276: 1291-1298Crossref PubMed Scopus (32) Google Scholar. Thus, neither the R-domain phosphorylation nor the C-terminal extension appears to be exclusively responsible for the failure to mature. Mutations identified in patients with cystic fibrosis are distributed all over the CFTR gene. Examination of more than 40 missense mutations indicated that a large proportion (60%) of those in cytoplasmic loops caused misprocessing, while none in the extracytoplasmic loops had this effect5.Seibert F.S. Loo T.W. Clarke D.M. Riordan J.R. Cystic fibrosis: Channel, catalytic, and folding properties of the CFTR protein.J Bioenerg Biomembr. 1997; 29: 429-442Crossref PubMed Scopus (37) Google Scholar. Hence, detection appears to occur on the cytoplasmic rather than the luminal side of the ER membrane. Consistent with this interpretation, although nascent CFTR normally binds calnexin, the conformation-sensing mechanism that this chaperone provides together with UDP-glucosyl glucose transferase, does not seem to be involved since prevention of glycosylation neither interferes with transport of wild-type CFTR to the cell surface nor enables δF508 to do so. The cytoplasmic chaperones Hsp70 and Hsp90 bind nascent CFTR and inhibition of the latter interaction with ansamycin drugs further disrupts maturation and accelerates proteolysis6.Loo M.A. Jensen T.J. Cui L. et al.Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (290) Google Scholar. As a Hsp90 substrate, nascent CFTR is typical of a small set of proteins that mature with difficulty. Because missense mutations at nearly all locations along the polypeptide may prevent maturation, it seems likely that achievement of a native overall tertiary structure is the criterion that must be met to enable export from the ER. Sequence changes elsewhere in the molecule can suppress or compensate for the null export phenotype of δF508. An example is the combined substitutions R29K and R555K that rescue at least one third of δF508 molecules7.Chang X. Cui L. Hou Y. et al.Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis.Mol Cell. 1999; 4: 137-142Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar.

Superiority of secondary hydroxy side modification in cyclodextrin complexation for hghly hydrophilic adenine nucleotides

Monoamino derivatives of β-cyclodextrin (β-CD) having an amino group either at the primary ( 1 ) or secondary hydroxy group side ( 2 ) were employed for investigating the superiority of the secondary hydroxy side modification to the guest binding of adenine nucleotides. Both 1 and 2 could form more stable host-guest complexes with AMP, ADP, and ATP than β-CD due to the electrostatic interaction between the ammonium group of the hosts and the phosphate groups of the guests. The effectiveness of the electrostatic interaction was supported by destabilization exerted by the presence of Mg 2+. The guest binding constants of 2 for AMP, ADP, and ATP were 79, 140, and 160 dm 3 mol −1, respectively, the values being larger than those of 1 by a factor of ∼2. On the other hand, comparable binding abilities between 1 and 2 were observed for adenosine incapable of interacting with a cationic ammonium group. These results demonstrate that 2 is a better host for the adenine nucleotides than 1 , and, combining our previous results on crown ether-modified β-CDs, indicate the superiority of the secondary hydroxy side modification upon molecular recognition for hydrophilic guest compounds by CDs.

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

The structure of the F(1)-ATPase from spinach chloroplasts was determined to 3.2 A resolution by molecular replacement based on the homologous structure of the bovine mitochondrial enzyme. The crystallized complex contains four different subunits in a stoichiometry of alpha(3)beta(3)gammaepsilon. Subunit delta was removed before crystallization to improve the diffraction of the crystals. The overall structure of the noncatalytic alpha-subunits and the catalytic beta-subunits is highly similar to those of the mitochondrial and thermophilic subunits. However, in the crystal structure of the chloroplast enzyme, all alpha- and beta-subunits adopt a closed conformation and appear to contain no bound adenine nucleotides. The superimposed crystallographic symmetry in the space group R32 impaired an exact tracing of the gamma- and epsilon-subunits in the complex. However, clear electron density was present at the core of the alpha(3)beta(3)-subcomplex, which probably represents the C-terminal domain of the gamma-subunit. The structure of the spinach chloroplast F(1) has a potential binding site for the phytotoxin, tentoxin, at the alphabeta-interface near betaAsp(83) and an insertion from betaGly(56)-Asn(60) in the N-terminal beta-barrel domain probably increases the thermal stability of the complex. The structure probably represents an inactive latent state of the ATPase, which is unique to chloroplast and cyanobacterial enzymes.

Subunit Interactions of Yeast NAD+-specific Isocitrate Dehydrogenase

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.

Allosteric Interactions between the Two Non-equivalent Nucleotide Binding Domains of Multidrug Resistance Protein MRP1

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.

Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy: nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1.

To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulfonylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, including a pair of siblings from a consanguineous family. We also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regulatory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mutations were found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K(ATP) function were evaluated using 86Rb+ efflux studies in COS-7 cells. SUR1-446fsdelT and SUR1-1436Q did not form a functional K(ATP). Western blot analysis after transient expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cells. K(ATP)(SUR1-1420C) showed reduced responses to metabolic inhibition by oligomycin and 2-deoxyglucose. K(ATP) channels are under complex regulation by intracellular ATP and ADP. ATP both inhibits and activates these channels. The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be through a regulatory subunit SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in regulating the K(ATP) channel. In SUR1-1420C, high-affinity binding of ATP to the nucleotide-binding fold (NBF)-1 was indistinguishable from that of wild-type SUR1. However, stabilization of ATP binding to NBF-1 by MgATP or MgADP was impaired, suggesting that this defect may account for impaired K(ATP)(SUR1-1420C) function. This is the first direct biochemical evidence that the cooperativity of nucleotide binding to SUR1 is impaired in a SUR1 mutant causing PHHI. No mutations were identified in the Kir6.2 and glutamate dehydrogenase genes. The genetic etiology of PHHI appears to be heterogeneous. SUR1 mutations may account for no more than 20% of PHHI cases in Japanese patients. Mutations of Kir6.2 and glutamate dehydrogenase genes are likely to be even less common.

2.9 Å crystal structure of ligand‐free tryptophanyl‐tRNA synthetase: Domain movements fragment the adenine nucleotide binding site

The crystal structure of ligand-free tryptophanyl-tRNA synthetase (TrpRS) was solved at 2.9 A using a combination of molecular replacement and maximum-entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl-5'AMP complex (TAM; Doublié S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17-31). In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal alphaA-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189-1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the "induced-fit" mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581-1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants.

Nucleotide Binding Drives Conformational Changes in the Isolated α and β Subunits of the F1-ATPase from Escherichia coli

The modeling of the rotatory mechanism performed by the F1-ATPase complex during ATP synthesis shows that the β, but not the α subunit, undergoes large conformational changes that depend on the occupancy of the catalytic site. Here we determined by fluorescence spectroscopy the changes in tertiary structure and hydrophobic exposed area of the isolated α and β subunits of the F1-ATPase complex from Escherichia coli upon adenine nucleotide binding. The results show that in the absence of intersubunit contacts, the two subunits exhibit markedly similar conformational movements.

Functional analysis of affinity-purified polyhistidine-tagged DnaA protein.

DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein.

Engineering of the Myosin-Iβ Nucleotide-binding Pocket to Create Selective Sensitivity to N6-modified ADP Analogs

Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosin's nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N(6) substituents. Introduction of this mutation, Y61G in rat myosin-Ibeta, did not alter the enzyme's affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N(6)-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N(6)-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Ibeta; in doing so, these analogs locked Y61G myosin-Ibeta tightly to actin. N(6)-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Ibeta. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Ibeta. Introduction of Y61G myosin-Ibeta molecules into a cell should permit selective inhibition by N(6)-modified ADP analogs of cellular processes dependent on myosin-Ibeta.