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• W1988756997 abstract "The nucleotide binding to uncoupling protein (UCP-1) of brown adipose tissue is regulated by pH. The binding pocket of the nucleotide phosphate moiety has been proposed to be controlled by the protonization of a carboxyl group (pK ≈4.5) for both nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) (identified as Glu-190) and of a histidine (pK ≈7.2) for NTP only. Here we identify His-214 as a pH sensor specific for NTP binding only. In reconstituted UCP-1 from hamster, DEPC diminishes binding of NTP but not of NDP. It also prevents inhibition of H+ transport by NTP but not by NDP. Hamster UCP-1 expressed in Saccharomyces cerevisiae was mutated to H214N resulting in only moderate change of the binding affinity for NTP (GTP) but a 10-fold affinity decrease with the bulkier substituent in H214W, whereas the affinity for NDP (ADP) was largely unchanged. The steep decrease with pH of the binding affinity for NTP in wild type (from pH 6.0 to 7.5) was much flatter in the mutants. Also, the pH dependence of binding and dissociation rates was diminished in these mutants. The transport of H+ and Cl− was not affected. Thus, His-214 is only involved in nucleotide binding, whereas, as previously shown, His-145 and His-147 are involved only in H+ transport. The results validate the earlier proposal of a histidine regulating the NTP binding in addition to a carboxyl group controlling both NTP and NDP binding. It is proposed that His-214 protrudes into the binding pocket for the γ-phosphate thus inhibiting NTP binding and that His214H+ is retracted by a background –CO2− group to give way for the γ-phosphate. The nucleotide binding to uncoupling protein (UCP-1) of brown adipose tissue is regulated by pH. The binding pocket of the nucleotide phosphate moiety has been proposed to be controlled by the protonization of a carboxyl group (pK ≈4.5) for both nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) (identified as Glu-190) and of a histidine (pK ≈7.2) for NTP only. Here we identify His-214 as a pH sensor specific for NTP binding only. In reconstituted UCP-1 from hamster, DEPC diminishes binding of NTP but not of NDP. It also prevents inhibition of H+ transport by NTP but not by NDP. Hamster UCP-1 expressed in Saccharomyces cerevisiae was mutated to H214N resulting in only moderate change of the binding affinity for NTP (GTP) but a 10-fold affinity decrease with the bulkier substituent in H214W, whereas the affinity for NDP (ADP) was largely unchanged. The steep decrease with pH of the binding affinity for NTP in wild type (from pH 6.0 to 7.5) was much flatter in the mutants. Also, the pH dependence of binding and dissociation rates was diminished in these mutants. The transport of H+ and Cl− was not affected. Thus, His-214 is only involved in nucleotide binding, whereas, as previously shown, His-145 and His-147 are involved only in H+ transport. The results validate the earlier proposal of a histidine regulating the NTP binding in addition to a carboxyl group controlling both NTP and NDP binding. It is proposed that His-214 protrudes into the binding pocket for the γ-phosphate thus inhibiting NTP binding and that His214H+ is retracted by a background –CO2− group to give way for the γ-phosphate. uncoupling protein brown adipose tissue fatty acid N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide 2′-O-[5-(dimethylamino)-naphthaline-1-sulfonyl]-GTP n-decylpentaoxyethylene wild type phenylmethanesulfonyl fluoride diethylpyrocarbonate 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid. The uncoupling protein (UCP-1)1 from brown adipose tissue short circuits H+/OH− generated by the respiratory chain, thus releasing heat from the oxidation of substrates (1Nicholls D.G. Biochim. Biophys. Acta. 1979; 549: 1-29Crossref PubMed Scopus (268) Google Scholar, 2Klingenberg M. Trends Biochem. Sci. 1990; 15: 108-112Abstract Full Text PDF PubMed Scopus (241) Google Scholar). The H+/OH− transport activity of UCP is regulated by fatty acids as activators and by purine ribose nucleotides as inhibitors (1Nicholls D.G. Biochim. Biophys. Acta. 1979; 549: 1-29Crossref PubMed Scopus (268) Google Scholar, 3Locke R.M. Rial E. Scott I.D. Nicholls D.G. Eur. J. Biochem. 1982; 129: 373-380Crossref PubMed Scopus (101) Google Scholar). The inhibition by nucleotides (ATP, ADP, GTP, and GDP) is further regulated by the H+concentration. With higher pH, the K I values for H+ transport inhibition and theK D values for nucleotide binding increase (4Klingenberg M. Biochemistry. 1988; 21: 2950-2956Google Scholar, 5Huang S.-G. Klingenberg M. Biochemistry. 1995; 34: 349-360Crossref PubMed Scopus (57) Google Scholar). This affinity decrease with pH is stronger with nucleoside triphosphates (GTP and ATP) than with the diphosphates. From the detailed analysis of the pK D/pH relation, a model was derived in which two H+ dissociating groups in the binding pocket of UCP are controlling the nucleotide binding (4Klingenberg M. Biochemistry. 1988; 21: 2950-2956Google Scholar, 5Huang S.-G. Klingenberg M. Biochemistry. 1995; 34: 349-360Crossref PubMed Scopus (57) Google Scholar). It was proposed that the protonation of an acidic residue (Glu or Asp) is prerequisite for binding both NDP and NTP and in addition the protonation of a His residue for NTP only. The dual pH control of NTP binding was regarded to be critical for relieving nucleotide binding within a narrow pH increase and thus to activate H+transport. Recently, we have been able to verify the existence of a –CO2H group as a pH sensor for nucleotide binding by using Woodward reagent K. This group was identified as Glu-190, which is located in the fourth transmembrane helix of UCP (6Winkler E. Wachter E. Klingenberg M. Biochemistry. 1997; 36: 148-155Crossref PubMed Scopus (46) Google Scholar). Furthermore, by using an expression system for UCP in Saccharomyces cerevisiae, we demonstrated that by mutagenic substitution of Glu-190 the affinity of nucleotide binding increased and became largely pH-independent (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). Thus the role of Glu-190 as a pH sensor for nucleotide binding in UCP was established. This encouraged the search for a His residue as a second pH sensor. Here we report the identification by mutagenesis of His-214 as a critical group participating in the pH control of NTP binding. The results provide evidence that sterical factors play a major role in the pH-dependent differentiation between nucleoside triphosphate versus diphosphate binding. In the neutral form His-214, suggested to be located in the binding pocket of the γ-phosphate, is concluded to protrude into the binding pocket, whereas in the charged state it is retracted by a background negative charge thus making space for the γ-phosphate. This pH-controlled movement of the His-214 is superimposed by the pH control of Glu-190 to produce the strong pH dependence of the nucleoside triphosphate binding, which should be of great importance for the regulation of the uncoupling activity of UCP. n-Decylpentaoxyethylene (C10E5), diethylpyrocarbonate, and Dowex 1-X8 (200–400 mesh) were obtained from Fluka. [14C]GTP, [14C]ADP, and [3H]GDP were from Amersham Corp. 2′-O-Dansyl-GTP was synthesized as described by Huang and Klingenberg (5Huang S.-G. Klingenberg M. Biochemistry. 1995; 34: 349-360Crossref PubMed Scopus (57) Google Scholar). The fluorescence dyes MQAE and pyranine (8-hydroxpyrene-1,3,6-trisulfonic acid, trisodium salt) were purchased from Molecular Probes. The gene coding for UCP-1 from hamster was cloned in pEMBLyE4 vector (8Cesareni G. Murry J.A.H. Seltow J.K. Genetic Engineering: Principles and Methods. 9. Plenum Publishing Corporation, New York1987: 135-154Crossref Google Scholar) under the control of the gal10/cyc1 promoter as described previously (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar, 9Bienengraeber M. Echtay K.S. Klingenberg M. Biochemistry. 1998; 37: 3-8Crossref PubMed Scopus (98) Google Scholar). The histidine mutant was generated by using an oligonucleotide-directed system (USE Mutagenesis Kit, Pharmacia). The CAT codon for His-214 was changed to the AAC and TGG codon for aspargine and tryptophan to construct H214N and H214W, respectively. The sequence of the mutant was verified by DNA sequencing, also checking for the absence of any other mutation in the UCP-coding frame. The S. cerevisiae strain W303 was transformed with a plasmid containing the mutation. Yeast transformants were grown in selective lactate medium, and expression was induced by adding 0.4% galactose. Mitochondria were isolated from yeast by differential centrifugation following a procedure previously described (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). Mitochondria were suspended in a solution containing 0.6 mmannitol, 20 mm Tris, pH 7.4, containing 0.5 mm EDTA, 0.1 mm EGTA, and 1 mmPMSF. Quantification of UCP incorporated in yeast mitochondria was performed by dansyl-GTP fluorescence titration. To remove endogenous residual bound nucleotide, mitochondria were treated with Dowex as mentioned previously (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). UCP was isolated from yeast mitochondria using Triton X-100 as detergent according to the protocol described for hamster brown adipose tissue mitochondria (10Lin C.S. Klingenberg M. FEBS Lett. 1980; 113: 299-303Crossref PubMed Scopus (213) Google Scholar) with modification as reported previously (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). Purified UCP was reconstituted into phospholipid vesicles following a protocol previously described (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). Briefly, UCP was suspended with egg yolk phosphatidylcholine (phospholipid:protein = 500:1 by mass), a medium of 100 mm potassium phosphate, pH 7.6, for H+ transport or 100 mm sodium phosphate, pH 6.8, for Cl− transport measurement in addition to 0.2 mm EDTA and 1 mm PMSF, and C10E5 (detergent:phospholipid = 1.4 by mass). Vesicle formation was accomplished by slow removal of the detergent with Bio-Beads SM-2 at 4 °C. The external solute was removed by passing the vesicle over a G-75 column. Vesicles for Cl− transport measurement were loaded with MQAE through diffusion for 17 h in the dark at 4 °C. UCP was isolated from mitochondria of brown fat adipose tissue and reconstituted into phospholipid vesicles as described previously (11Klingenberg M. Winkler E. Huang S.-G. Methods Enzymol. 1995; 260: 369-389Crossref PubMed Scopus (22) Google Scholar). For H+uptake measurements, vesicles were loaded with 100 mmpotassium phosphate and 0.4 mm EDTA, pH 7.5. Vesicles with a phospholipid concentration of 19 and 0.124 mg/ml protein were treated with increasing concentrations of DEPC (0 to 2.2 mm) resulting in DEPC:His ratio of 0–150 mol/residue. After modification by shaking for 1 h at 8 °C, the external solutes were removed from the proteoliposomes by Sephadex G-75 column, and the H+ transport measurements were performed as described (11Klingenberg M. Winkler E. Huang S.-G. Methods Enzymol. 1995; 260: 369-389Crossref PubMed Scopus (22) Google Scholar) using a low impedance combination pH electrode. For UCP expressed in yeast, H+ uptake activity was measured on an MPF-44A fluorescence spectrophotometer using pyranine fluorescence at λex = 467 nm and λem = 510 nm in a standard medium containing 280 mm sucrose, 0.5 mm Hepes, 0.2 mm EDTA, 1 μm pyranine, pH 6.9, and 125 μm lauric acid. Valinomycin (2.5 μm) was added to initiate the K+ gradient-driven H+uptake. Cl− transport was measured by fluorescence of MQAE-loaded proteoliposome at λex = 355 nm and λem = 460 nm in a medium containing 4 mmsodium phosphate, pH 6.8, and 155 mm KCl. Cl−influx was initiated by the addition of valinomycin (2 μm), and the rate (J CL) was determined using a two-point calibration method as described by Verkmanet al. (12Verkman A.S. Takla R. Sefton B. Basbaum C. Widdicombe J.H. Biochemistry. 1989; 28: 4240-4244Crossref PubMed Scopus (45) Google Scholar) as shown in Equation 1.JCL=[(Fi−Fs)(KCl+1/[Cl]s)]−1[dF(0)/dt](Eq. 1) The applied methods for transport measurements resembled those described previously (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). Binding titration with [14C]GTP and [14C]ADP followed in principle the published procedure using Dowex for removal of free radiolabeled nucleotide (4Klingenberg M. Biochemistry. 1988; 21: 2950-2956Google Scholar, 7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). The binding rate of [14C]GTP was measured with an automated rapid mixing and separating sampling machine developed in our laboratory as described previously (7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). Nucleotide fluorescence derivative (dansyl-GTP) was applied for GTP dissociation rate determination. The dissociation was followed by the fluorescence decrease on addition of large excess ATP after fluorescence equilibrium of dansyl-GTP binding to UCP at λex = 350 nm and λem = 525 nm. The buffer used was 20 mm Hepes or Mes at 15 °C. For elucidating the proposed role of histidine in nucleotide binding, we applied the amino acid reagent diethyl pyrocarbonate (DEPC), which is reported to react primarily with histidine (13Miles E.W. Methods Enzymol. 1977; 47: 431-442Crossref PubMed Scopus (815) Google Scholar). In experiments with hamster BAT mitochondria, the reagent inhibited nucleotide binding only at very high concentrations where also reactions with primarily amino groups and —SH groups occur. With isolated UCP, DEPC at a critical concentration of 80 mol/mol His present in UCP decreases the binding of GTP to 60% and of GDP to 85% (Fig. 1). However, at higher concentration no difference on the effect of DEPC can be seen between di- and triphosphate. The inhibition of nucleotide binding by DEPC increased strongly with the pH, indicating that the neutral form of His reacts with DEPC, in agreement with reports with other enzymes (14Brandsch M. Brandsch C. Ganapathy M.E. Chew C.S. Ganapathy V. Leibach F.H. Biochim. Biophys. Acta. 1997; 1324: 251-262Crossref PubMed Scopus (44) Google Scholar, 15Lim J. Turner A.J. FEBS Lett. 1996; 381: 188-190Crossref PubMed Scopus (11) Google Scholar, 16Bragg P.D. Hou C. Eur. J. Biochem. 1996; 241: 611-618Crossref PubMed Scopus (15) Google Scholar). In the reconstituted system the effect of DEPC could be measured both on H+ transport and its inhibition by nucleotides as well as on the nucleotide binding. The results in Fig. 1 show that DEPC only minimally affects the H+ translocation activity of UCP at up to 150 mol of DEPC/mol of His present in UCP. The inhibition of H+transport by GDP or by GTP is differently influenced by DEPC. Whereas the inhibition by GDP is decreased by DEPC only from 86 to 78%, DEPC decreases dramatically the ability of GTP to inhibit to only 10%. Thus the interference of DEPC with the nucleotide interaction is highly selective for GTP as compared with GDP. In parallel binding measurements, GTP binding is found to be lowered with DEPC by 60%, whereas GDP binding is only marginally affected. These results indicate that histidine is involved specifically with the binding of GTP in accordance with the predictions. The use of dansyl-GTP titration allows quantification of H214N and H214W UCP incorporation into mitochondria which is estimated to about 2.5% of the total protein content similar to that of wt. Fig. 2 shows the SDS gel and immunoblot of isolated UCP and mitochondria, respectively. The isolated proteins from wt and His-214 mutant UCP are largely freed from two main contaminants, namely porin and ADP/ATP carrier. The yield of isolated wt and H214W UCP is 1.2–1.5% of total mitochondrial protein, but only 0.5–0.7% for H214N UCP which is half the amount obtained as compared with wt. The purity of the isolated wt and H214W protein is estimated to about 70% based on maximum binding of [14C]GTP in comparison to maximum binding of highly purified UCP from hamster. Since the binding capacity of the isolated H214N UCP is 40% less (as shown below), the purity of the isolated protein is estimated the same as the wt based on SDS gel comparison. H+uptake in proteoliposomes reconstituted with purified UCP requires a K+ diffusion potential negative inside, generated by the valinomycin-catalyzed efflux of K+ and high buffering capacity inside. Cl− uptake is induced by a K+diffusion potential positive inside, generated by the valinomycin-induced influx of K+. The rates of H+ and Cl− uptake are summarized in Fig. 3. Proteoliposomes reconstituted with H214W transport H+ and Cl− at the same rate as wt, but with H214N only to about 60% of wt. Treatment of the proteoliposomes with 100 μm GTP or GDP leads to 85% inhibition of transport activities for both wt and His-214 mutant UCP. The residual activity may be ascribed to inversely inserted UCP molecules, whose binding sites cannot be accessed by the nucleotides. The 40% lower transport activity of H214N mutant UCP is in line with the same percentage decrease in nucleotide binding capacity. Thus, the turnover of H214N in H+ and Cl− transport is the same as wt UCP. We can conclude that His-214 is not involved in the transport activities of UCP. To study the binding to isolated UCP of nucleoside tri- and diphosphate and its pH dependence, titration with [14C]GTP and [14C]ADP using the Dowex method (see “Experimental Procedures”) was performed at different pH values. GDP had to be replaced by ADP, as a purine nucleoside diphosphate, since no [14C]GDP available. The mass action plot evaluated from the concentration dependence with [14C]GTP (Fig. 4) gives aB max = 12 μmol/g of protein of GTP bound for wt, 15 μmol/g of protein for H214W, and 8 μmol/g of protein for H214N UCP. Thus the binding capacity is increased by 25% in H214W and reduced by 40% in H214N as compared with wt on isolation of UCP, whereas the binding determined for mitochondria is the same in the mutants and wt. A similar percentage variation is obtained with [14C]ADP. Table I lists theK D values of several binding experiments and titrations at pH 6.0, 6.8, and 7.5 for both [14C]GTP and [14C]ADP. The K D for [14C]GTP of wt increases 8-fold from 1.05 μm at pH 6.8 to 7.9 μm at pH 7.5, whereas in His-214 mutants a slight change in the affinity is observed with only 1.5-fold increase of K D in H214N and almost no variation in H214W from pH 6.8 to 7.5. In contrast to the low pH sensitivity shown with nucleoside triphosphate (GTP), the affinity of His-214 mutant to nucleoside diphosphate (ADP) is highly pH-sensitive. As shown in Table I and Fig. 4, the K D value for [14C]ADP increases by shifting the pH from 6.0 to 7.5 in H214W by the same factor, i.e, ≈12 times as in wt and ≈18 times in H214N.Table IDissociation constant (KD) of wild-type, H214N, and H214W UCP-1 by [C]GTP and [C]ADP at different pHUCPpH[14C]GTPK D[14C]ADPK DμmμmWild type6.00.391.056.81.053.167.57.9013.0H214N6.00.740.796.83.003.17.54.7014.6H214W6.05.80.976.89.42.467.59.512.5The K D values were evaluated from [14C]GTP titration of 200 μg/ml UCP on ice.The K D values were evaluated from [14C]ADP titration of 200 μg/ml UCP on ice. Open table in a new tab The K D values were evaluated from [14C]GTP titration of 200 μg/ml UCP on ice. The K D values were evaluated from [14C]ADP titration of 200 μg/ml UCP on ice. In order to elucidate further the involvement of His-214 on the pH regulation of nucleoside triphosphate binding, a kinetic study of [14C]GTP binding was performed. Binding and dissociation rate determinations supplement the equilibrium measurements and can reflect changes in affinity under conditions, e.g. low or very high affinity, where directK D determinations are difficult. Table II summarizes the rate constants (k on) evaluated from the initial slope according to the second-order reaction at different pH values. Thek on for the wt UCP decreases about 7-fold, from pH 6.5 to 7.5. For the two His-214 mutants, on the other hand, the time progress curves (Fig. 5) of binding clearly illustrate the slower binding at low pH and the faster binding rate at high pH as compared with wt. The k on of H214N and H214W changes slightly (1.4-fold) from pH 6.5 to 7.5 as compared with a 7-fold decrease in the case of wt.Table IIRates of [C]GTP binding and dansyl-GTP dissociation of wild-type, H214N, and H214W UCP-1 at different pH and 15 °CUCPpHk on × 10−3k off, × 10−3mssWild type6.00.254.96.50.2012.77.50.0356.0H214N6.00.167.16.50.0914.57.50.0722.3H214W6.00.112.76.50.078.87.50.0511.1k on were evaluated from time study of [14C]GTP binding to UCP at 15 °C.k off were evaluated from the fluorescence decrease on addition of large excess of ATP after binding of dansyl-GTP to isolated UCP protein at 15 °C. Open table in a new tab k on were evaluated from time study of [14C]GTP binding to UCP at 15 °C. k off were evaluated from the fluorescence decrease on addition of large excess of ATP after binding of dansyl-GTP to isolated UCP protein at 15 °C. For measuring the dissociation rates, the fluorescent nucleotide derivative (dansyl-GTP) was used and evaluated from the fluorescence decrease upon removal of dansyl-GTP on addition of large excess ATP. Table II lists the dissociation rates (k off) for wt, H214N, and H214W UCP at different pH values. Thek off with wt UCP increases about 5-fold from pH 6.5 to 7.5, whereas with H214N and H214W thek off changes just about 1.4-fold. Remarkably, the dissociation rate with H214W is lower than with wt and H214N at low pH. The pH dependence of the binding and dissociation rate constants are plotted in Fig. 6. As measured with [14C]GTP, the binding rate for wt UCP decreases with pH according to Δpk on/ΔpH ≈1, and the dissociation rate of dansyl-GTP increases with pH according to Δpk off/ΔpH ≈ −1. For H214N and H214W UCP, the k on and k offchange less between pH 6.5 and 7.5, resulting in almost zero slope of Δpk on/ΔpH and Δpk off/ΔpH. The regulation of the uncoupling activity of UCP is an important factor in the control of thermogenesis. The major players are fatty acids as activators and purine nucleotides as inhibitors of H+ transport by UCP. In addition, the variation of pH influences the nucleotide effect. In BAT cells even in the activated state the concentration of the inhibiting free ATP4− at pH 7.0 may still largely exceed the low K I and K D for this nucleotide. Only by increasing the pH above 7.0, the affinity for nucleotides may be sufficiently decreased for generating unliganded UCP (4Klingenberg M. Biochemistry. 1988; 21: 2950-2956Google Scholar). A model of the intracellular regulation will be suggested below. Interestingly, this affinity decrease expressed as ΔpK D/ΔpH is much steeper (−2) with nucleoside triphosphates (GTP and ATP) than (−1) with diphosphates (GDP and ADP) so that with increasing pH, UCP may be rapidly relieved from the most abundant inhibitor ATP4−. This pH control was visualized to be due to two H+-dissociating groups at the nucleotide binding center of UCP, one –CO2H group common for both NDP and NTP and an additional group with a pK ≈7.2, e.g. His for NTP only. It was proposed that only the additional positive charge provided by HisH+ is necessary for binding of NTP4− but not for NDP3−. The –CO2H group pH sensor was previously identified as Glu-190 (6Winkler E. Wachter E. Klingenberg M. Biochemistry. 1997; 36: 148-155Crossref PubMed Scopus (46) Google Scholar, 7Echtay K.S. Bienengraeber M. Klingenberg M. Biochemistry. 1997; 36: 8253-8260Crossref PubMed Scopus (47) Google Scholar). It should be noted that out of the four His occurring in UCP, His-214 as well as His-145 and His-147 are conserved among UCP from all sources known so far. His-145 and His-147 have been found by mutagenesis to be involved in H+ translocation, but their replacement did not affect nucleotide binding and its pH dependence (9Bienengraeber M. Echtay K.S. Klingenberg M. Biochemistry. 1998; 37: 3-8Crossref PubMed Scopus (98) Google Scholar). Thus only His-214 is left as a candidate for pH sensor of NTP binding. Indeed, here by mutagenesis the involvement of His-214 in nucleotide binding and its pH regulation are shown, although in a somewhat different version than predicted. Both identifications vindicate the original model derived from the pH dependence of nucleotide binding. These results contradict the nondocumented claims by Mordriansky et al. (17Mordriansky M. Murdza-Inglis D.L. Patel H.V. Freeman K.B. Garlid K.D. J. Biol. Chem. 1997; 272: 24759-24762Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) that no histidine and specifically no His-214 is involved in nucleotide binding. The finding reported here that the His reagent DEPC inhibits binding of GTP but not of GDP and concomitantly that DEPC desensitizes H+ transport against the inhibition by GTP but not by GDP provides evidence for the involvement of a histidine residue in nucleoside triphosphate binding only. The fact that H+transport is unaffected by DEPC although both His-145 and His-147 have been previously shown to be involved specifically in H+transport points to a preferred sensitivity of His-214 toward DEPC. This suggests, in line with our model, that His-214 is located in the hydrophilic binding pocket for the γ-phosphate, where the hydrophilic DEPC can easily enter and due to a high resident time reacts preferably with His-214. In contrast, His-145 and His-147 are either not accessible or are on the surface where DEPC cannot reach a critical concentration. According to the original model, replacement of His by a neutral group should drastically lower the binding affinity for the nucleoside triphosphates. However, the binding affinity of GTP is found to persist in H214N but is much weaker in H214W. With increasing pH, the affinity decreases much less in H214N and H214W than in wt UCP. The mutations, however, do not change the binding affinity for ADP (which is used here to replace unavailable [14C]GDP) showing the same pH dependence as in wt UCP. Similarly, the rates of binding and dissociation as measured with [14C]GTP and fluorescent dansyl-GTP lose their strong pH dependence in the His-214 mutants. As a result of the low pH dependence, the affinity for GTP is somewhat lower at pH 6.8 in His-214 mutants and especially in H214W than in wt but is distinctly higher at pH 7.5. This crossover is also clearly seen in the pH dependence of the binding and dissociation rates. The slower dissociation rate of fluorescent (dansyl-GTP) nucleotide observed in H214W versus H214N seems to contradict the lower affinity of His-214 as measured with [14C]GTP. Possibly the dansyl derivative of GTP has an additional interaction with the introduced Trp side chain. The substitution of His-214 by aspargine lowers the stability of UCP upon isolation from mitochondria, and thus the content of intact UCP is about 40% of wt as determined by GTP binding, whereas on substitution by tryptophan, the protein retains its stability. Correspondingly, the capacity for transporting H+ and Cl− is decreased by about 40% in the H214N and is retained completely in H214W mutant. In relation to the GTP-binding capacity as a measure of intact UCP, the transport activities in H214N are unchanged as compared with wt. Thus, the results with both mutants show that His-214 is not involved in H+ and Cl− transport. This agrees with the broad evidence that the nucleotide-binding site is not in the translocation channel (18Klingenberg M. Biochem. Soc. Trans. 1984; 12: 390-393Crossref PubMed Scopus (25) Google Scholar, 19Rial E. Nicholls D.G. Eur. J. Biochem. 1986; 161: 689-694Crossref PubMed Scopus (20) Google Scholar, 20Kopecky J. Jezek P. Drahota Z. Houstek J. Eur. J. Biochem. 1987; 164: 687-694Crossref PubMed Scopus (19) Google Scholar, 21Huang S.-G. Klingenberg M. Biochemistry. 1996; 35: 7846-7854Crossref PubMed Scopus (29) Google Scholar). His-214, located at the c side of the 5th transmembrane helix, seems to be close to the previously established pH sensor Glu-190, located at the c side of the 4th helix. Thus His-214 as well as Glu-190 could be located in the binding pocket for the phosphate moiety of the nucleotide. According to the original model, a His (now His-214) should be primarily in contact with the γ-phosphate, whereas a –CO2H (now Glu-190) should be at the gate for accessing both nucleoside di- and triphosphate (Fig. 7). Furthermore, HisH+ was assumed to form an ion bond with γ-phosphate. The marked decrease of the pH dependence of nucleoside triphosphate but not diphosphate binding is in line with the role of His-214 as a pH sensor. However, the retention of NTP binding in His-214 mutants requires a change of our model. At low pH, in H214N, the affinity is only slightly lower than in wt UCP, indicating that HisH+ does not contribute to NTP binding. It now seems that His-214 in the neutral form, abundant at pH > 7 repulses NTP, since the replacement of His by Asn increases affinity at high pH. Our new model, as illustrated in Fig. 7, retained the assumption that His is located at the end of the binding pocket. It shows that the imidazole of the neutral His protrudes into the pocket, thus sterically restraining the binding of triphosphate. On protonization, the HisH+ group is retracted by a background –CO2− group, thus leaving space for the γ-phosphate. In H214N the smaller Asn side chain does not restrain γ-phosphate binding, and the pH control is largely lost. In H214W the bulkier side chain may at least partially protrude into the binding pocket as judged from the 9-fold lower binding affinity. In agreement with this model, the His-DEPC derivative, which is unable to accept a H+ and has its hydrophilic extension, would protrude into the binding pocket at any pH and thus block NTP but not NDP binding. In conclusion, out of the four His only His-214 essentially fulfills the criteria of an additional pH controller specific for NTP binding. Together with the previously identified pH sensor Glu-190, it provides an additional pH regulation of the H+ transport activity of UCP. Both work by changes of electrostatic interaction on H+ binding. Glu-190 as a gatekeeper at the entrance to the phosphate binding pocket, common to NDP and NTP, opens the pocket on protonation, and His-214 makes space for the γ-phosphate of NTP when being retracted as HisH+ by a negative background charge. How can the strong pH dependence of NTP binding be incorporated into a consistent picture of the regulation of uncoupling in BAT cells? The major binding nucleotide to UCP can be assumed to be ATP. In the resting state, at high Mg2+ and at pH ≈7, the free ATP4−:ADP3− ratio is still high despite the lower affinity of ADP3− for Mg2+. Thus, UCP may be well saturated by ATP4−, and evidence for this has been presented by showing that UCP in mitochondria isolated from short term warm-adapted BAT is masked to a large extent by ATP (22Huang S.-G. Klingenberg M. Eur. J. Biochem. 1995; 229: 718-725Crossref PubMed Scopus (29) Google Scholar). LaNoueet al. (23LaNoue K.F. Koch C.D. Meditz R.B. J. Biol. Chem. 1982; 257: 13740-13748Abstract Full Text PDF PubMed Google Scholar, 24LaNoue K.F. Strzelecki T. Strzelecka D. Koch C. J. Biol. Chem. 1986; 261: 298-305Abstract Full Text PDF PubMed Google Scholar) made an analysis of the uncoupling regulation in BAT cells and presented impressive arguments in favor of a control by ATP. They estimated that due to the small volume of cytosol in BAT, a small load on ATP would drastically lower the cytosolic ATP concentration. The norepinephrine-induced FA release and ensuing FA activation would generate this ATP load. In addition, lowering the Δψ on redistribution of ATP into mitochondria contributes to the decrease of cytosolic ATP. The results also suggest that FA somehow modulates the ATP effect on UCP (24LaNoue K.F. Strzelecki T. Strzelecka D. Koch C. J. Biol. Chem. 1986; 261: 298-305Abstract Full Text PDF PubMed Google Scholar). On the other hand, it has been shown by us (25Winkler E. Klingenberg M. J. Biol. Chem. 1994; 269: 2508-2515Abstract Full Text PDF PubMed Google Scholar) that there is no competition of FA with nucleotide binding. Therefore we argue that the putative FA effect on ATP inhibition has to be identified with a pH increase to >7.2. After norepinephrine stimulation, mitochondria are flooded with free FA. Their uptake into the matrix can cause a cytosolic pH increase which is becoming more important since the ratio of the cytosol to mitochondrial volume is small. In fact, a 0.8 unit increase of the cytosolic pH relative to mitochondria has been determined on strong load with FA in rat liver perfused with octanoate (26Soboll S. Gründel S. Schwabe U. Scholz R. Eur. J. Biochem. 1984; 141: 231-236Crossref PubMed Scopus (42) Google Scholar). In addition to lowering the affinity to UCP, the pH increase lowers the ATP4− concentration in favor of ATP-Mg2+formation. Furthermore, as shown by Huang et al. (27Huang S.-G. Lin Q.-S. Klingenberg M. J. Biol. Chem. 1998; 273: 859-864Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), the increase of pH strongly facilitates the otherwise strikingly low dissociation of ATP from UCP. At this point we cannot exclude other mechanisms of pH increase, such as signaling by FA of pH regulation units, e.g. the Na+/H+ exchanger, either directly by FA or by AMP which is drastically increased in the cytosol. This mechanism of the uncoupling regulation in BAT is at variance with that proposed by Nicholls and co-workers (28Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1353) Google Scholar, 29Rial E. Poustie A. Nicholls D.G. Eur. J. Biochem. 1983; 137: 197-203Crossref PubMed Scopus (149) Google Scholar) who assume that nucleotide binding and dissociation do not play a role in the acute regulation but that in vivo only FA regulates the conductivity of UCP to H+. Nucleotides are assumed to be permanently bound and to shift the conductivity/voltage relation to a nonlinear dependence. Uncoupling occurs with a break potential of 150–170 mV. However, the measurements on isolated BAT indicate that with norepinephrine Δψ decreases to about 60 mV (24LaNoue K.F. Strzelecki T. Strzelecka D. Koch C. J. Biol. Chem. 1986; 261: 298-305Abstract Full Text PDF PubMed Google Scholar), that is into a range where conductivity of UCP with bound nucleotide would be very low. It is noteworthy that in the new UCP members, UCP-2 and UCP-3, both Glu-190 and His-214 are conserved, although the homology to UCP-1 is only 59% in UCP-2 and 57% in UCP-3 (30Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.E. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-273Crossref PubMed Scopus (1562) Google Scholar, 31Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.-P. FEBS Lett. 1997; 408: 39-41Crossref PubMed Scopus (998) Google Scholar, 32Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (682) Google Scholar). Based on the present results, this indicates that in UCP-2 and UCP-3, nucleotide binding and its pH control follow similar principles as found here for UCP-1. In contrast, His-145 and His-147, previously identified to participate in the H+ transport of UCP-1, are not conserved in UCP-2 and UCP-3, indicating a variant mechanism of fatty acid-dependent H+ transport. The advice from Dr. Shu-Gui Huang on the use of fluorescent nucleotide derivative and the help of Ilse Prinz for the preparation of mitochondria are gratefully acknowledged." @default.
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