A Cl and Cl NMR study of chloride binding to the erythrocyte anion transport protein

Band 3, the erythrocyte anion transport protein, mediates the one-for-one exchange of bicarbonate and chloride ions across the membrane and consequently plays an important role in respiration. Binding to the protein forms the first step in the translocation of the chloride across the membrane. ³⁵Cl and ³⁷Cl NMR relaxation measurements at various field strengths were used to study chloride binding to the protein in the presence and absence of the transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate. Significant differences occurred in the NMR relaxation rates depending on whether the inhibitor was present or not. The results indicate that the rate of chloride association and dissociation at each external binding site occurs on a time scale of 5 μs. This implies that the transmembrane flux is not limited by the rate of chloride binding to the external chloride binding site of band 3. The rotational correlation-time of chloride bound to band 3 was found to be 20 ns with a quadrupole coupling constant of 3 MHz.     

Evidence that anion transport by band 3 proceeds via a ping-pong mechanism involving a single transport site. A 35 Cl NMR study

Band 3 catalyzes the one-for-one exchange of monovalent anions across the red cell membrane. At least two anion binding sites have been postulated to exist on the transport unit: 1) a transport site that has been observed by saturation kinetics and by 35 Cl NMR studies of chloride binding, and 2) a 35Cl NMR-invisible inhibitory site that has been proposed to explain the inhibition of anion exchange at large anion concentrations. A number of independent studies have indicated that the transport site is alternately exposed to different sides of the membrane during the transport cycle. Yet the role, if any, of the postulated inhibitory site in the transport cycle is not known. Here it is shown that: 1) when the [Cl-], [Br-], or pH is varied, the band 3 transport sites on both sides of the membrane behave like a homogeneous population of simple anion binding sites in 35Cl NMR experiments, and 2) when the [Cl-] is varied, the outward-facing transport site behaves like a simple anion binding site. These results indicate that the postulated inhibitory site has no effect on chloride binding to the transport site. Instead, the results are quantitatively consistent with the ping-pong model (Gunn, R. B., and Fr?lich, O. (1979) J. Gen. Physiol. 74, 351-374), which states that the transport site is the only site involved in the transport cycle. Expressions are derived for the macroscopically observed characteristics of a ping-pong transporter: these characteristics are shown to be weighted averages of the microscopic properties of the inward- and outward-facing conformations of the transport site. In addition to supporting the simplicity of the transport mechanism, the high pH titration curve for chloride binding to the transport site provides insight into the structure of the site. The macroscopically observed pKA = 11.1 +/- 0.1 in the leaky ghost system indicates that an arginine must provide the essential positive charge in the inward- or outward-facing conformation of the transport site, or in both conformations.     

Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance.

We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.     

The minimal structure containing the band 3 anion transport site. A 35Cl NMR study

35Cl NMR, which enables observation of chloride binding to the anion transport site on band 3, is used in the present study to determine the minimal structure containing the intact transport site. Removal of cytoskeletal and other nonintegral membrane proteins, or removal of the 40-kDa cytoskeletal domain of band 3, each leave the transport site intact. Similarly, cleavage of the 52-kDa transport domain into 17- and 35-kDa fragments by chymotrypsin leaves the transport site intact. Extensive proteolysis by papain reduces the integral red cell membrane proteins to their transmembrane segments. Papain treatment removes approximately 60% of the extramembrane portion of the transport domain and produces small fragments primarily in the range 3-7 kDa, with 5 kDa being most predominant. Papain treatment damages, but does not destroy, chloride binding to the transport site; thus, the minimal structure containing the transport site is composed solely of transmembrane segments. In short, the results are completely consistent with a picture in which the transport site is buried in the membrane where it is protected from proteolysis; the transmembrane segments that surround the transport site are held together by strong attractive forces within the bilayer; and the transport site is accessed by solution chloride via an anion channel leading from the transport site to the solution.     

Conformational selection by the aIF2 GTPase: a molecular dynamics study of functional pathways

Archaeal initiation factor 2 (aIF2) is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To improve our understanding of the role of each conformational state in the aIF2 "life cycle", we start from the state immediately after GTP hydrolysis, ON:GDP:P(i) (where P(i) is inorganic phosphate), and consider the possible next steps on the pathway to the OFF:GDP product. The first possibility is P(i) dissociation, leading to ON:GDP, which could then relax into OFF:GDP. We use molecular dynamics simulations to compute the P(i) dissociation free energy and show that dissociation is highly favorable. The second possibility is conformational relaxation into the OFF state before P(i) dissociation, to form OFF:GDP:P(i). We estimate the corresponding free energy approximately, 2 ± 3.5 kcal/mol, so that this is an uphill or weakly downhill process. A third possibility is relaxation into another conformation, neither ON nor OFF. Indeed, a third, "MIXED" conformation was seen recently in a crystal structure of the aIF2:GDP:P(i) complex. For this conformational state, P(i) dissociation is weakly unfavorable, in contrast to the ON and OFF states. From this, we will deduce that if the MIXED:GDP complex is not too unstable, the ON:GDP:P(i) → MIXED:GDP:P(i) transformation is a downhill process, which can occur spontaneously. This suggests that the MIXED state could be a functional intermediate.     

DNA-binding properties of gene-5 protein encoded by bacteriophage M 13. 1. The kinetics of the dissociation of gene-5-protein.polynucleotide complexes upon addition of salt

The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein.poly(dA) complexes and for the gene-5-protein.M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein.M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel is not maintained. The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide. The absence of line broadening for gene-5-protein.polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.     

Methods for measuring rates of protein binding to insoluble scaffolds in living cells: histone H1-chromatin interactions

Understanding of cell regulation is limited by our inability to measure molecular binding rates for proteins within the structural context of living cells, and many systems biology models are hindered because they use values obtained with molecules binding in solution. Here, we present a kinetic analysis of GFP-histone H1 binding to chromatin within nuclei of living cells that allows both the binding rate constant k(ON) and dissociation rate constant k(OFF) to be determined based on data obtained from fluorescence recovery after photobleaching (FRAP) analysis. This is accomplished by measuring the ratio of bound to free concentration of protein at steady state, and identifying the rate-determining step during FRAP recovery experimentally, combined with mathematical modeling. We report k(OFF) = 0.0131/s and k(ON) = 0.14/s for histone H1.1 binding to chromatin. This work brings clarity to the interpretation of FRAP experiments and provides a way to determine binding kinetics for nuclear proteins and other cellular molecules that interact with insoluble scaffolds within living cells.     

p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes

The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N-ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p-(chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport.     

Mechanism of competition between chloride and stilbenedisulfonates for binding to human erythrocyte band 3 (AE1).

Stilbenedisulfonates (S) constitute an important class of competitive inhibitors of the anion exchange (AE) function found in plasma membranes of various cell types. I present a brief summary of recent kinetic studies that provide insight into the mechanism of stilbenedisulfonate-chloride competition in binding to human erythrocyte band 3 (AE1) (B), the chloride-bicarbonate exchanger. Reversible stilbenedisulfonate binding follows a two-step mechanism (S + B <--> SB <--> SB*). Several lines of evidence are summarized that show that chloride, stilbenedisulfonates, and band 3 form a ternary complex, with chloride lowering stilbenedisulfonate affinity allosterically, by accelerating the rate of stilbenedisulfonate release. Of particular significance was our evidence demonstrating that extracellular chloride could accelerate stilbenedisulfonate release from its binding site on the outer surface of band 3 in resealed ghosts (i.e., acceleration in the release of a bound competitive inhibitor by a cis substrate). I suggest that the latter result may be consistent with our earlier proposal that band 3 follows a two-site ordered sequential mechanism, where two allosterically linked chloride binding transport sites move back and forth across the membrane together.     

Halide-selective,proton-coupled anion transport by phenylthiosemicarbazones

Phenylthiosemicarbazones (PTSCs) are proton-coupled anion transporters with pH-switchable behaviour known to be regulated by an imine protonation equilibrium. Previously, chloride/nitrate exchange by PTSCs was found to be inactive at pH 7.2 due to locking of the thiourea anion binding site by an intramolecular hydrogen bond, and switched ON upon imine protonation at pH 4.5. The rate-determining process of the pH switch, however, was not examined. We here develop a new series of PTSCs and demonstrate their conformational behaviour by X-ray crystallographic analysis and pH-switchable anion transport properties by liposomal assays. We report the surprising finding that the protonated PTSCs are extremely selective for halides over oxyanions in membrane transport. Owing to the high chloride over nitrate selectivity, the pH-dependent chloride/nitrate exchange of PTSCs originates from the rate-limiting nitrate transport process being inhibited at neutral pH.     

Molecular mechanisms of band 3 inhibitors. 1. Transport site inhibitors

The band 3 protein of red cells is a transmembrane ion transport protein that catalyzes the one-for-one exchange of anions across the cell membrane. 35Cl NMR studies of Cl- binding to the transport sites of band 3 show that inhibitors of anion transport can be grouped into three classes: (1) transport site inhibitors (examined in this paper), (2) channel-blocking inhibitors (examined in the second of three papers in this issue), and (3) translocation inhibitors (examined in the third of three papers in this issue). Transport site inhibitors fully or partially reduce the affinity of Cl- for the transport site. The dianion 4,4'-di-nitrostilbene-2,2'-disulfonate (DNDS) and the arginine-specific reagent phenylglyoxal (PG) each completely eliminate the transport site 35Cl NMR line broadening, and each compete with Cl- for binding. These results indicate that DNDS and PG share a common inhibitory mechanism involving occupation of the transport site: one of the DNDS negative charges occupies the site, while PG covalently modifies one or more essential positive charges in the site. In contrast, 35Cl NMR line broadening experiments suggest that 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) leaves the transport site partially intact so that the affinity of Cl- for the site is reduced but not destroyed. This result is consistent with a picture in which DIDS binds near the transport site and partially occupies the site.     

Two-stage binding of SecA to the bacterial translocon regulates ribosome-translocon interaction

The bacterial translocon interacts with both SecA-bound preproteins and nascent chain-ribosome complexes during Sec and signal recognition particle-dependent protein translocation, respectively. In their inactive state, translocons are saturated with ribosomes and SecA protein, reflecting the inherent affinity of these components for one another. We found that SecA and ribosomes are bound simultaneously and noncompetitively to a common set of inactive translocons. Furthermore, we demonstrate that at a later stage in binding, SecA possesses a ribosome-translocon dissociation activity that is coupled to its ATP-dependent membrane insertion and retraction cycle that drives protein translocation. This novel activity is presumably important in the commitment of the translocon to the Sec-dependent pathway. These results also provide a rationale for the compatibility and regulation of multiple protein translocation pathways that each makes distinct demands on a common translocon core.     

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and P_i release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and P_i. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.     

A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis

The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group. Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton. Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important. The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane. Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm. After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.     

The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.     

Direct observation of the transmembrane recruitment of band 3 transport sites by competitive inhibitors. A 35Cl NMR study

Numerous models describing anion exchange across the red cell membrane by band 3 have been discussed in literature. These models are readily distinguished from one another by an experiment which tests the ability of band 3 transport sites to be recruited to one side of the membrane. In order to observe directly the transmembrane recruitment of transport sites, we have developed 35Cl NMR techniques that resolve the two transport site populations on opposite sides of the membrane. Using these techniques, we show that the inhibitors 4,4'- dinitrostilbene -2,2'-disulfonate and p- nitrobenzensulfonate each recruit all of the transport sites on both sides of the membrane to the extracellular facing conformation. This result indicates that band 3 has an alternating site transport mechanism: each band 3 transport unit possesses a single functional transport site which is alternately exposed first to one side of the membrane then to the other.     

The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.     

Nigericin-mediated H+, K+ and Na+ transports across vesicular membrane: T-jump studies.

The decay of delta pH across vesicular membranes by nigericin-mediated H+ and metal ion (M+) transports has been studied at 25 degrees C after creating delta pH by temperature jump (T-jump). In these experiments K+ or Na+ were chosen as M+ for the compensating flux. Theoretical expressions derived to analyse these data suggest a method for estimating the intrinsic rate constants for the translocation of nig-H (k1) and for the translocation of nig-M (k2) across membrane, from the pH dependence of the delta pH decay. The following could be inferred from the analysis of data. (a) At pH approximately 7.5 and 250 mM ion concentrations, nigericin-mediated H+ and M+ transport rates are lower in a medium of K+ than in a medium of Na+, although ionophore selectivity of nigericin towards K+ is 25-45-times higher than that towards Na+. However, at lower [M+] (approximately 50 mM) the transport rates are higher in a medium of K+ than in a medium of Na+. Such behaviours can be understood with the help of parameters determined in this work. (b) The intrinsic rate constants k1 and k2 associated with the translocations of nig-H and nig-K or nig-Na across membrane are similar in magnitude. (c) At pH approximately 7.5 translocation of nig-H is the dominant rate-limiting step in a medium containing K+. In contrast with this, at this pH, translocation of nig-M is the dominant rate-limiting step when metal ion is Na+. (d)k1 approximately k2 approximately 6.10(3) s-1 could be estimated at 25 degrees C in vesicles prepared from soyabean phospholipid, and lipid mixtures of 80% phosphatidylcholine (PC) + 20% phosphatidylethanolamine and 92% PC + 8% phosphatidic acid. (e) The apparent dissociation constants of nig-M in vesicles were estimated to be approximately 1.5.10(-3) M for K+ and 6.4.10(-2) M for Na+ (at 50 mM ion concentrations) using approximately 10(-8.45) M for the apparent dissociation constant of nig-H.     

31P and 35Cl nuclear magnetic resonance measurements of anion transport in human erythrocytes

The exchange of anions across the erythrocyte membrane has been studied using 31P nuclear magnetic resonance (NMR) to monitor inorganic phosphate influx and 35Cl NMR to monitor chloride ion efflux. The 31P NMR resonances for intracellular Pi and extracellular Pi could be observed separately by adjusting the initial extracellular pH to 6.4, while the intracellular pH was 7.3. The 35Cl NMR resonance for intracellular Cl- was so broad as to be virtually undetectable (line width greater than 200 Hz), while that of extracellular Cl-is relatively narrow (line width of about 30 Hz). The transports of Pi and Cl-were both totally inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, a potent inhibitor of the band 3 protein. Since the 31P resonance of Pi varies with pH, intra- and extracellular pH changes could also be determined during anion transport. The extracellular pH rose and intracellular pH fell during anion transport, consistent with the protonated monoanionic H2PO4-form of Pi being transported into the erythrocyte rather than the deprotonated dianionic HPO24-form. The rates of Cl-efflux and Pi influx were determined quantitatively and were found to be in close agreement with values determined by isotope measurements. The Cl-efflux was found to coincide with the influx of the monoanionic H2PO4-form of Pi.