Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis

Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for halorhodopsin. In buffer containing nitrate, halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two pigments. At the extracellular end of the presumed helix C, however, an arginine residue is found in halorhodopsin, but not in pharaonis halorhodopsin, and an arginine-rich segment between the presumed helices A and B in halorhodopsin is replaced by a less positively charged sequence in pharaonis halorhodopsin (Lanyi, J. K., Duschl, A., Hatfield, G. W., May, K., and Oesterhelt, D. (1990) J. Biol. Chem. 265, 1253-1260). One or both of these alterations may explain the difference in the anion selectivity of the two proteins.     

Effects of anion binding on the deprotonation reactions of halorhodopsin

The retinal Schiff base of halorhodopsin deprotonates with a pKa of 7.4 in 0.5 M Na2SO4 in the dark. In the presence of various anions, such as chloride or nitrate, etc., the pKa is raised by up to 1.5 units. Analysis of the dependency of the pKa on anion concentration favors the model in which the anions do not bind to the positively charged Schiff base nitrogen, but to a site near it, and exert their effect on the pKa by direct (perhaps electrostatic) interaction. Adding nitrate, or one of several other anions, causes also a small blueshift in the visible absorption band of the chromophore. These effects on the pKa and the absorption band define an anion binding site in halorhodopsin, termed Site I. Chloride and bromide apparently bind in addition to another site, which is associated with a small red-shift of the absorption band and changes in the photocycle. This other anion binding site is termed Site II. Illumination of halorhodopsin samples results in the deprotonation of the Schiff base with a much lowered pKa, but at very low rates probably determined by the generation of a deprotonating photointermediate. Binding of Site I anions increases the pKa of deprotonation in the light also. The similarity of the responses of the apparent pKa in the dark and in the light to anion concentration suggests that anion binding to Site I influences deprotonation of the Schiff base similarly in the photointermediate and in the parent halorhodopsin molecule.     

Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism.

The light-driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison.     

Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.     

Position-dependent effects of polylysine on Sec protein transport

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.     

Light-dependent trans to cis isomerization of the retinal in halorhodopsin

Flash-induced absorption changes in the near UV were determined for bacteriorhodopsin and halorhodopsin on a millisecond time scale. The difference spectrum obtained for bacteriorhodopsin was comparable to model difference spectra of tyrosine (aromatic OH deprotonated vs protonated), as found by others. The flash-induced difference spectrum for halorhodopsin, in contrast, resembled a model spectrum obtained for trans to 13-cis isomerization of retinal in bacteriorhodopsin. A model for chloride translocation by halorhodopsin is presented, in which the retinal isomerization moves positive charges, which in turn modulate the affinity of a site to chloride.     

Direct evidence that the proton motive force inhibits membrane translocation of positively charged residues within membrane proteins

The M13 phage procoat protein requires both its signal sequence and its membrane anchor sequence in the mature part of the protein for membrane insertion. Translocation of its short acidic periplasmic loop is stimulated by the proton motive force (pmf) and does not require the Sec components. We now find that the pmf becomes increasingly important for the translocation of negatively charged residues within procoat when the hydrophobicity of the signal or membrane anchor is incrementally reduced. In contrast, we find that the pmf inhibits translocation of the periplasmic loop when it contains one or two positively charged residues. This inhibitory effect of the pmf is stronger when the hydrophobicity of the inserting procoat protein is compromised. No pmf effect is observed for translocation of an uncharged periplasmic loop even when the hydrophobicity is reduced. We also show that the Delta Psi component of the pmf is necessary and sufficient for insertion of representative constructs and that the translocation effects of charged residues are primarily due to the DeltaPsi component of the pmf and not the pH component.     

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface

The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14-20 mutations. Generally, the tightest binding variants showed similar associated rate constants (k(on)) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (DeltaCp) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone-receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process.     

In vitro kinetic analysis of the role of the positive charge at the amino-terminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in Escherichia coli

By using an in vitro system for the translocation of secretory proteins in Escherichia coli, detailed and quantitative studies were performed as to the function of the positively charged amino acid residues at the amino terminus of the signal peptide. Uncleavable OmpF-Lpp, a model secretory protein carrying an uncleavable signal peptide, and mutant proteins derived from it were used as translocation substrates. When the positive charge, +2 (LysArg) for the wild-type, was changed to 0, -1, or -2, little or no translocation was observed. The number of the positive charge was altered by introducing different numbers of Lys or Arg residues into the amino terminus. The rate of translocation was roughly proportional to this number, irrespective of whether the charged amino acid residues were Lys or Arg. When the amino-terminal LysArg was replaced by His residues, translocation took place more efficiently at pH 6.5 than pH 8.0, whereas that of the wild-type was about the same as the two pH values. We conclude that the signal peptide requires a positive charge at its amino-terminal region to function in the translocation reaction and that the rate of translocation is roughly proportional to the number of the positively charged group, irrespective of the amino acid species that donates the charge. Evidence suggesting that the positive charge is involved in the binding of precursor proteins to the membrane surface to initiate translocation is also presented.     

Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR

Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 +/- 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F-, citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride-transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site. We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites.     

Charged Amino Acids in a Preprotein Inhibit SecA-Dependent Protein Translocation

Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA.     

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group a protein on interaction with DNA intermediates of nucleotide excision repair

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the inter-action of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E ? K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.     

Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids

The effects of positively and negatively charged residues on the membrane topology of a model E. coli protein with two transmembrane segments have been studied. We show that addition or removal of as little as a single positively charged lysine residue in one of two critical regions can be sufficient to reverse the transmembrane topology of the molecule from Nout-Cout to Nin-Cin. Negatively charged residues are much less potent and significantly affect the topology only if present in high numbers. Finally, we provide data to suggest that sec-independent and sec-dependent translocation mechanisms differ in their sensitivity to positively charged amino acids.     

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.     

Properties and the primary structure of a new halorhodopsin from halobacterial strain mex.

A new halorhodopsin-like pigment from the new halobacterial strain mex (Otomo, J., Tomoika, H. and Sasabe, H. (1992) J. Gen. Microbiol. 138, 1027-1037) was partially purified, and its amino acid sequence from helices A to G was determined using PCR technique. Two arginine residues in the A-B interhelix loop segment, a series of six amino acid residues (EMPAGH) in the B-C interhelix segment and most of the residues near the Schiff base of the retinal were found to be conserved in three halorhodopsins (halobium, pharaonis and mex). This result strongly suggests that these residues are essential for anion pumping function in halorhodopsin. The light-induced ion-pump measurements have shown that the selectivity of anion transport between chloride and nitrate in mex halorhodopsin is lower than that of halobium halorhodopsin, but higher than that of pharaonis halorhodopsin. The number of amino acid residues in the B-C interhelix loop segments is different in each halorhodopsin, and it correlates with their anion (chloride and nitrate) selectivity. These results suggest that the length of the B-C segment affects the selectivity of anion transport in halorhodopsin.     

Relating structure to thermodynamics: the crystal structures and binding affinity of eight OppA-peptide complexes.

The oligopeptide-binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine-X-Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X-ray crystallography. The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain. Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems. The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects. Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge. Marked entropy-enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.     

The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

The role of the membrane electrochemical potential in the translocation of acidic and basic residues across the membrane was investigated with the M13 procoat protein, which has a short periplasmic loop, and leader peptidase, which has an extended periplasmically located N-terminal tail. For both proteins we find that the membrane potential promotes membrane transfer only when negatively charged residues are present within the translocated domain. When these residues are substituted by uncharged amino acids, the proteins insert into the membrane independently of the potential. In contrast, when a positively charged residue is present within the N-terminal tail of leader peptidase, the potential impedes translocation of the tail domain. However, an impediment was not observed in the case of the procoat protein, where positively charged residues in the central loop are translocated even in the presence of the membrane potential. Intriguingly, several of the negatively charged procoat proteins required the SecA and SecY proteins for optimal translocation. The studies reported here provide insights into the role of the potential in membrane protein assembly and suggest that electrophoresis can play an important role in controlling membrane topology.     

Dual Mechanism of Ion Permeation through VDAC Revealed with Inorganic Phosphate Ions and Phosphate Metabolites

In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a “charged brush” which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.