Decrease In Apparent Affinity Research Articles

Structure‐Function Studies of the Thermotoga maritima ADPGlucose Pyrophosphorylase: Probing the role of the C‐terminus of the GlgD subunit

ADP Glucose pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step of glycogen and starch biosynthesis in plants and bacteria. Structure‐function studies of the enzyme from different sources will allow the engineering of a more active enzyme to produce more renewable and biodegradable carbon. In Thermotoga maritima (T.ma) there are two genes, glgC and glgD, which code for two subunits of this ADPG PPase. The recombinant His‐tagged subunits were successfully purified using a heat step and nickel chromatography. Previous studies performed at 37ºC (pH 7.5) had shown that the wild type (WT) glgD alone had no activity, while the glgC protein displayed a specific activity of ~ 3 Unit/mg with S0.5 values for ATP and magnesium (Mg2+) of 5.39 mM and 18.66 mM, respectively. However, the WT glgC/D complex at subsaturating substrate conditions was stimulated ~20‐fold compared to glgC alone and was also activated ~2‐fold by fructose‐1,6‐ bisphosphate (FBP). The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. In the presence of FBP, the apparent affinity for ATP increased ~6‐fold for the complex with a modest ~30% increase in Vmax. Previous alignment studies and molecular modeling principally focused on the functional role of the N‐terminus. Therefore the truncation of 73 amino acids at the C‐terminal end of glgD was generated to test its potential functional role in the complex. In the absence of FBP, the truncated complex exhibited a modest decrease in ATP apparent affinity for ATP and Mg2+ with ~20‐fold and ~2 fold decreased in Vmax, respectively, compared to the WT complex. It was noted that in the presence of the truncated glgD, the Vmax was substantially lower than for glgC alone, indicating an inhibitory role. In the presence of FBP, the truncated complex displayed a ~6‐fold decrease in apparent affinity for ATP, ~3‐fold for Mg and a ~6‐fold lower Vmax compared to the WT complex. However, the Vmax of the truncated complex is close to the Vmax of the glgC alone indicating that FBP diminishes the inhibition by the truncated glgD. The primary effect of the truncation appears to be on the Vmax of the complex as well as a reduction in the K‐type activation by FBP observed with the WT. Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of WT GlgC/WT GlgD are still underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Probing the role of the N‐terminus in the ADP‐Glucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

Glucans serve as critical energy storage compounds in nearly all living organisms. Such renewable and biodegradable carbon sources are in demand now from industry as an eco‐friendly substitute for petroleum based products. ADP‐Glucose Pyrophosphorylase (ADPG PPase) is the enzyme that catalyzes the rate limiting step of glucan biosynthesis in plants and bacteria, respectively. The novel Thermodesulfovibrio yellowstonii (Td.y) ADPG PPase, from a thermophilic, sulfate‐reducing bacterium, has potential properties that could lead to engineering a very stable and highly active form which could increase the yield of starch. Further, sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N‐terminal regions (glycine rich and RRAKPAV) that are known to be important for regulation and catalysis in other characterized ADPG PPases. The recombinant Wild‐type (WT) and variants (E15A, F23R, S25A) have been successfully purified by hydroxyapatite and size exclusion chromatography. In the case of E15A, preliminary data displayed similar velocity and similar apparent substrate affinity for ATP compared to WT, in the absence of effector molecules. However, in the presence of the activator PEP, E15A shows a ~7 fold increase in activity compared to 4 fold in WT. This suggests a role in regulation and binding of effector molecules. In the absence of effector molecules, preliminary data from F23R reveal a 4 fold decrease in activity and a ~2 fold decrease in apparent affinity for ATP compared to the WT. However, in the presence of effector molecules, ATP affinity was restored to WT values. In the presence of PEP and FBP, F23R displays a ~7 fold increase in activation compared to WT. The F23 position appears to play a role in structure or folding/allosteric regulation, which is also supported by molecular modeling. These results suggest a potential for altered binding of negatively charged metabolites due to increased positively charged residues in the postulated binding pocket. Kinetic data for the variant S25A reveals a Vmax ~3 fold higher than WT with a10‐fold higher apparent affinity for ATP compared to the WT as well as desensitization to effector molecules. This position in Td.y may play a role in setting the relative activity level that can be further attenuated by effectors. Along with kinetic analyses, initial crystallization trials are underway for variant S25A with initial crystals from different conditions diffracting to a resolution of ~3.96 Å. Results gathered from the WT and variants thus far confirm and extend the role of the two conserved regions for activity and regulation.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Structure‐function studies of ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii: Probing the Role of the C‐terminus in Allosteric Regulation

Starch serves as both a critical food source and an important biodegradable and renewable carbon source for biofuel and other industries including adhesives, packaging material, and cosmetics. ADP‐glucose pyrophosphorylase (ADPG PPase, glgC gene product) is the key enzyme that is responsible for the rate‐limiting step of both glycogen and starch biosynthesis in bacteria and plants, catalyzing the conversion of glucose‐1‐phosphate and ATP to ADPG and pyrophosphate. Thermodesulfovibrio yellowstonii (Td. y) is a thermophilic sulfate‐reducing bacterium that was isolated from geothermal vents in Yellowstone National Park. Little is known about this ADPG PPase which displays increased heat stability, salt tolerance, and altered regulation compared to other ADPG PPase family members. The Td.y enzyme is substantially activated by phosphoenolpyruvate (PEP), which differs from many other ADPG PPases, as well as G6P, 3PGA, and sulfate. These properties could contribute to biomass production in different crop environments. Multiple sequence alignments have shown deviations from consensus residues in the N‐terminal allosteric region and C‐terminal region of the Td.y ADPG PPase. Two of the more interesting differences are at positions 365 and 397, which have been shown to be involved in allosteric regulation of other ADPG PPases, as part of a conserved region in the enzyme family and associated with plant enzymes, respectively. The K365G and R397A enzymes were generated by site‐directed mutagenesis as a first step in probing structure‐function relationships. We hypothesize that the positively charged lysine at 365 and arginine at 397 may contribute to PEP binding and activation. The altered proteins were purified to homogeneity by a procedure that included two hydroxyapatite chromatography steps followed by size exclusion chromatography. Initial kinetic studies revealed that the K365G protein displayed a lower Vmax (~25% of wild‐type [WT]) but ~2‐fold higher apparent affinity for ATP in the absence of effectors. In contrast to the WT, which was activated over 3‐fold by PEP, the K365G enzyme only exhibited ~10% activation by PEP and a decrease in apparent affinity for ATP. Conversely, the K365G enzyme was activated 2.6 fold by sulfate, an effector which has a much smaller effect on the Vmax of WT. In the absence of effectors, the R397A displayed a similar apparent affinity for ATP compared to WT but a 24 fold decrease in Vmax. In the presence of PEP, R397A exhibited lower apparent affinity for ATP and a Vmax decreased ~42 fold. The response of this enzyme to G6P was very similar to WT. The results so far support the hypothesis that K365 and R397 are critical for PEP activation while also influencing other allosteric properties. Complete kinetic analyses of the K365G and R397A enzymes as well as crystallization trials are underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Calcium and Pip2 Interplay Regulates BK Channel Activity via the RCK1 Gating Ring

BK channels are high conductance K+ channels that are activated by voltage and intracellular Ca2+. Ca2+ binds to sites in the RCK2 and RCK1 domains of Slo1, the pore-forming subunit of BK channels, to enhance voltage-dependent gating of the channel. PIP2, a ubiquitous modulator of ion channel activity, enhances Ca2+-driven gating of BK channels but a molecular understanding of this interplay remains elusive. Here, we report that amino acid residues in the RCK2 calcium bowl that bind Ca2+ do not affect PIP2 sensitivity. In contrast, the RCK1 Ca2+ coordination site is intimately involved in PIP2 sensitivity. We identified two critical residues within the alphaB helix, K392 and R393, are directly involved in coordinating PIP2. In the absence of Ca2+, the alphaB helix interacts with the alphaA helix to decrease the apparent affinity of the channel to PIP2 and inhibit channel activity. Ca2+ binding to the RCK1 site engaging D367 of the KDRDD loop relieves the decrease in PIP2 apparent affinity and channel inhibition. Furthermore, mutation of residues in the KDRDD loop or the alphaA/alphaB residues that couple this region to the alphaB PIP2 regulatory site also relieve the decrease in the apparent affinity for PIP2 and current inhibition in the absence of Ca2+. In addition, we show that the PMA-induced current inhibition also exhibits a dependence on channel-PIP2 interactions that determine the extent of modulation. Together these results show that along with Ca2+ and voltage, PIP2 is a critical third factor providing integral control of BK channel activity.

Abstract 282: Phospholamban Truncation Mutations Including Heart Failure Mutant L39stop Disrupt Membrane Localization.

Introduction: Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca 2+ handling and contractility by reversibly inhibiting SR Ca 2+ ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization. Hypothesis: We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation. Results and Methods: To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization. Conclusions: Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.

Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIPn), including phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP).Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIPn application. However, PIPn induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIPn-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIPn application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIPn regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIPn sensitivity to heteromeric channels formed with PIPn-insensitive A subunits. Finally, channels formed by mixtures of PIPn-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIPn regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels.

An Inverse Relationship Links Temperature and Substrate Apparent Affinity in the Ion-Coupled Cotransporters rGAT1 and KAAT1

The effects of temperature on the operation of two ion-coupled cotransporters of the SLC6A family, namely rat GAT1 (SLC6A1) and KAAT1 (SLC6A19) from Manduca sexta, have been studied by electrophysiological means in Xenopus laevis oocytes expressing these proteins. The maximal transport-associated current (Imax) and the apparent substrate affinity (K05) were measured. In addition to the expected increase in transport rate (Q10 = 3–6), both transporters showed greater K05 values (i.e., a decrease in apparent affinity) at higher temperatures. The transport efficiency, estimated as Imax/K05, increased at negative potentials in both transporters, but did not show statistically significant differences with temperature. The observation that the apparent substrate affinity is inversely related to the transport rate suggests a kinetic regulation of this parameter. Furthermore, the present results indicate that the affinities estimated at room temperature for mammalian cotransporters may not be simply extrapolated to their physiological operating conditions.

Evidence for Specific Roles of Arginines 26 and 38 in the Allosteric Regulation of ADP Glucose Pyrophosphorylase from Thermus thermophilus

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosteric enzyme that catalyzes the rate‐limiting step in glucan synthesis, an attractive target for engineering to increase the production of renewable carbon. The thermophillic bacterial enzyme from Thermus thermophilus was found to be activated by G6P, F6P, and FBP and displayed optimal activity at 75°C. Based on alignment and molecular modeling studies, R26 and R38 were proposed to be invovled in allosteric regulation of this enzyme. Altered proteins (R26A and R38A) were generated, expressed, and purified. R26A and R38A enzymes displayed a significant decrease in Vmax and a decrease in the apparent affinity for substrates compared to the wild‐type enzyme. In contrast to the wild‐type, the presence of activators caused an increased apparent affinity for the substrate ATP for both enzymes. The R26A mutation resulted in an ~8‐fold decrease in apparent affinity for G6P at 37°C, and a ~30‐fold decrease at 75°C. The R38A enzyme showed ~6‐fold and ~8‐fold decreases in apparent affinity for G6P and F6P at 37°C, respectively. Preliminary data shows decreased apparent affinity for substrates at 75°C for both enzymes. The diminution of the Vmax is consistent with a role for these arginines in stabilizing an active conformation of the enzyme. In addition, R26 appears to play a role in the binding of G6P while R38 appears to facilitate binding of both G6P and F6P in the allosteric subsite(s). Complete kinetic characterization of the altered enzymes and double mutant R26A, R38A at 75°C is underway. Supported by NSF Award 0448676.

Subunit-Specific Regulation of Photoreceptor CNG Channels by Phosphoinositides

Cyclic nucleotide gated (CNG) channels in retinal photoreceptor cells play a key role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric A3+B3 (cone) and A1+B1 (rod) channels are sensitive to regulation by PIP3 or PIP2, demonstrating a decrease in apparent affinity for cGMP. To determine what subunit types are necessary for PIP3 sensitivity, we generated heteromeric channels by co-expression of PIP3-insensitive A2ΔN (Brady et al., 2006) with B3 or B1 subunits.

Spectroscopic Investigation of Phosphorylated Pentameric Phospholamban Bound to the Sarcoplasmic Calcium ATPase

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is the main enzyme responsible for the reuptake of Ca2+ into the sarcoplasmic reticulum (SR) during muscle relaxation. Phospholamban (PLN), a homopentameric membrane protein expressed in the SR, binds to and decreases the rate of Ca2+ transport of SERCA. This inhibitory action is relieved when PLN is phosphorylated at residues Ser-16 or Thr-17 in response to different signal pathways. Although a great deal of knowledge has been accumulated on the functional consequences of PLN phosphorylation, very little is known about the molecular details leading to relief of inhibition. Previous work suggested a crucial role of protein topology in the complex between SERCA and PLN, with the latter showing different tilt angles in the presence and absence of SERCA. Our hypothesis is that SERCA activity is regulated by changes in topology in PLN transmembrane helices upon phosphorylation. In order to probe for the orientation of protein domains, solid-state NMR in oriented lipid bilayers has established itself as the method of choice in many studies. Here we present data on the topological changes taking place in phospholamban pentamer in the presence of SERCA upon phosphorylation at Ser-16 or Thr-17. SERCA and PLN are reconstituted in lipid bilayers and uniaxially aligned either mechanically (on glass plates) or magnetically (upon formation of bicelles). The two proteins are fully functional under these conditions as demonstrated by the ability of SERCA to transport Ca2+ in functional assays as well as PLN dependent decrease in apparent affinity for Ca2+.

Ca2+-Induced Conformational Changes of Na+-Ca2+ Exchanger Dimers: Role of Ca2+ Binding Domains

The Na+-Ca2+ exchanger is activated by the binding of cytoplasmic Ca2+ to two Ca2+ binding domains (CBD1 and CBD2). How binding of Ca2+ is translated into exchanger activation is unknown. We investigated Ca2+-dependent movements as changes in FRET between exchanger dimers tagged with CFP or YFP at positions 266 within the large cytoplasmic loop of NCX1.1. The biophysical properties of the fluorescent exchangers are identical to those of the untagged NCX. Fluorescent exchangers were coexpressed in Xenopus oocytes from which plasma membrane sheets were isolated. Upon addition of Ca2+, the coexpressed pair NCX-266CFP + NCX-266YFP showed an increase in FRET in a dose-dependent manner. Similar FRET changes were observed after mutating the Ca2+ coordination site in CBD2 (E516L). Exchanger E516L is not Ca2+ regulated. In contrast, mutating the Ca2+ coordination site in CBD1 (D421A, E451A and D500V) abolished FRET changes. These residues likely disrupt binding of Ca2+ to CBD1. Nevertheless, Ca2+ regulation of NCX is retained though with a substantial decrease in apparent affinity for Ca2+. These results indicate that the Ca2+-induced conformational changes of NCX dimers arise exclusively from the movement of CBD1. Peptides of Ca2+ binding domains, flanked by CFP and YFP, recapitulated the full length exchanger results: CBD1 showed movement upon Ca2+ addition while CBD2 did not. A peptide spanning CBD1-CBD2 displayed Ca2+-dependent movement, which was abolished by mutating the Ca2+ coordination site in CBD1. Our results indicate the following: 1. Exchanger conformational changes are associated with the occupancy of a high affinity Ca2+ binding site exclusively within CBD1. 2. FRET studies confirm that the Na+-Ca2+ exchanger exists as a dimer.

Anticonvulsant and anxiolytic-like effects of compounds isolated from Polygala sabulosa (Polygalaceae) in rodents: in vitro and in vivo interactions with benzodiazepine binding sites

Polygala sabulosa, a folk medicine, presents dihydrostyryl-2-pyrones (DST) and styryl-2-pyrones (STY), compounds structurally similar to kavalactones. Our previous study showed that the ethyl acetate fraction (EA) and these constituents present anxiolytic-like, hypno-sedative, and anticonvulsant effects in mice. This study investigated the role of benzodiazepine binding site (BDZ-bs) in the central effects of either EA or three DST (1, 2, and 3) and three STY (4, 5, and 7), using in vivo and in vitro assays. In the elevated plus-maze (EPM), flumazenil (FMZ), a BDZ antagonist, partially blocked the anxiolytic-like effect of DST-3 or STY-4 and STY-7, but not DST-1. Using electroencephalogram (EEG), EA protected against pentylenetetrazole (PTZ)-induced convulsion in rats, an effect partially blocked by FMZ, suggesting the participation of the BDZ-bs in this action. EA also protected against the maximal electroshock (MES)-induced convulsions in mice, a profile distinct from diazepam (DZP). DST and STY compounds inhibited the [(3)H]-flunitrazepam ([(3)H]-FNZ) binding to BDZ-bs in rat cortical synaptosomes with K (i) higher than 100 microM (DST-1), 41.7 microM (DST-2), 35.8 microM (DST-3), 90.3 microM (STY-4), 31.0 microM (STY-5) and 70.0 microM (STY-7). In the saturation assay, DST-3 and STY-7 competitively inhibited the binding of [(3)H]-FNZ to BDZ-bs with a significant decrease in apparent affinity (K (d)) and no change in maximal binding (B (max)). The present data support a partial BDZ-bs mediation of the anxiolytic-like and anticonvulsant effects of EA of P. sabulosa and its main isolated constituents, DST and STY.

Mechanism of Reversal of Phospholamban Inhibition of the Cardiac Ca2+-ATPase by Protein Kinase A and by Anti-phospholamban Monoclonal Antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.

The Presence of Sarcolipin Results in Increased Heat Production by Ca2+-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.

Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.

Effects of mutations at conserved TM II residues on ligand binding and activation of mouse 5-HT 6 receptor

An aspartate residue (Asp-72) in the transmembrane helix II of mouse 5-hydroxytryptamine-6 receptor (5-HT 6) is conserved among most G protein-coupled receptors. We have examined the functional significance of this residue by site-directed mutagenesis. A single Asp → Ala (D72A) mutation resulted in an 8-fold decrease in apparent affinity for 5-HT, and a 60-fold reduction in EC 50 value of agonist-induced stimulation of adenylyl cyclase. A F69L/T70I/D72A triple mutant showed a 2-fold reduction in apparent affinity for 5-HT but complete loss of adenylyl cyclase stimulation. Binding of SB-258585 (4-iodo- N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]benzene-sulfonamide), a selective 5-HT 6 antagonist, was mildly affected (2- to 4-fold decrease in affinity) in the two mutants. Our data suggest that Asp-72 and additional residues toward the intracellular side of TM II have a limited role in ligand binding but are critical for functional activation of the 5-HT 6 receptor.

A Conserved Glutamate Residue in Transmembrane Helix 10 Influences Substrate Specificity of Rabbit OCT2 (SLC22A2)

OCT1 and OCT2 are involved in renal secretion of cationic drugs. Although they have similar selectivity for some substrates (e.g. tetraethylammonium (TEA)), they have distinct selectivities for others (e.g. cimetidine). We postulated that "homolog-specific residues," i.e. the 24 residues that are conserved in OCT1 orthologs as one amino acid and in OCT2 as a different one, influence homolog-specific selectivity and examined the influence on substrate binding of three of these conserved residues that are found in the C-terminal half of the rabbit orthologs of OCT1/2. The N353L and R403I substitutions (OCT2 to OCT1) did not significantly change the properties of OCT2. However, the E447Q replacement shifted substrate selectivity toward an OCT1-like phenotype. Substitution of glutamate with cationic amino acids (E447K and E447R) abolished transport activity, and the E447L mutant displayed markedly reduced transport of TEA and cimetidine while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the three-dimensional structure of OCT2, Glu(447) was found in a putative docking region within a hydrophilic cleft of the protein. In addition, six residues identified in separate studies as exerting significant effects on OCT binding were also found within the putative cleft region. There was a significant correlation (r(2) = 0.82) between the IC(50) values for inhibition of TEA transport by 14 different compounds and their calculated K(D) values for binding to the model of rabbit OCT2. The results suggest that homology modeling offers an opportunity to direct future site-directed studies of OCT/substrate interaction.

Functional characterization of non-synonymous single nucleotide polymorphisms in the gene encoding human multidrug resistance protein 1 (MRP1/ABCC1)

The 190-kDa ATP-binding cassette (ABC) multidrug resistance protein 1 (MRP1) encoded by the MRP1/ABCC1 gene mediates the active cellular efflux of glucuronide, glutathione and sulfate conjugates. It can also confer resistance to a diverse spectrum of chemotherapeutic agents and transport a variety of toxicants. In the present study, we examined 10 MRP1/ABCC1 missense genetic variants [non-synonymous single nucleotide polymorphisms (SNPs)] to determine whether or not they affect expression or function of the transporter. Variants 218C>T (Thr73Ile), 257C>T (Ser92Phe), 350C>T (Thr117Met), 689G>A (Arg230Gln), 1898G>A (Arg633Gln), 2168G>A (Arg723Gln), 2965G>A (Ala989Thr), 3140G>C (Cys1047Ser), 3173G>A (Arg1058Gln) and 4535C>T (Ser1512Leu) were recreated using site-directed mutagenesis and transfected into human embryonic kidney cells. Immunoblotting experiments showed that all mutant proteins were expressed at levels comparable to wild-type MRP1. Vesicular transport assays revealed that the Ala989Thr mutation caused a significant decrease in estradiol 17beta-glucuronide transport due to a decrease in apparent affinity (Km) for this organic anion. The transport properties of the other mutants were comparable to wild-type MRP1. When the MRP1/ABCC1 non-synonymous SNPs were evaluated by the SIFT algorithm using subsets of homologs and orthologs of MRP1/ABCC1, Arg230Gln, Val353Met, Arg433Ser, Gly671Val and Arg1058 mutations were predicted to be deleterious, whereas the PolyPhen algorithm predicted Ser92Phe and Gly671Val to be potentially damaging. Thus most predictions of these algorithms were not in accordance with our experimental results. In conclusion, our data suggest that none of the MRP1/ABCC1 variants studied are likely by themselves to have major deleterious effects in healthy individuals, and the SIFT and PolyPhen algorithms appear to be poor predictors of the phenotypic consequences of these MRP1 mutations at least in vitro.

Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5 ′-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5 ′-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5 ′-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a ∼15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.

How phospholamban could affect the apparent affinity of Ca 2+-ATPase for Ca 2+ in kinetic experiments

Binding of phospholamban (PLN) to the Ca 2+-ATPase of muscle sarcoplasmic reticulum results in a decrease in apparent affinity for Ca 2+ without affecting the true binding constant for Ca 2+ determined in equilibrium binding experiments. It is shown that this can be explained by a scheme in which the ATPase shows two modes of binding for PLN, one of high and one of low affinity; the proposed scheme is not dependent on the kinetic model assumed for the Ca 2+-ATPase.