Major transmembrane movement associated with colicin Ia channel gating

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin- containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.     

Topography of diphtheria Toxin's T domain in the open channel state

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a "double dagger" model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed "daggers," TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of approximately 70 residues, is translocated across the membrane to the trans side.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain

Summary C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments. Apparently trypsin removes segments of protein N-terminal to the channel-forming region, since gating behavior of the shortest fragment is little affected by the enzyme. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. Trypsin modifies only closed channels from thecis side (the side to which protein has been added) and only open channels from thetrans side. These results suggest that regions outside the channel-forming domain affect ion selectivity and gating, and they also provide evidence that large protein segments outside the channel-forming domain are translocated across the membrane with channel gating.     

Plug-and-Play Pairing via Defined Divalent Streptavidins

Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4 Å resolution and trans-divalent streptavidin to 1.6 Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.     

Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End?

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an approximately 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: the role of protein translocation

Summary The voltage-dependent channel formed in planar lipid bilayers by colicin E1, or its channel-forming C-terminal fragments, is susceptible to destruction by the nonspecific protease pepsin under well-defined conditions. In particular, pepsin acts only from thecis side (the side to which colicin has been added) and only upon channels in the closed state. Channels in the open state are refractory to destruction bycis pepsin, and neither open nor closed channels are destroyed bytrans pepsin. Colicin E1 channels are normally turned on bycis positive voltages and turned off bycis negative voltages. For large (>80 mV) positive voltages, however, channels inactivate subsequent to opening. Associated with the inactivated state, some channels become capable of being turned on bycis negative voltages and turned off bycis positive voltages, as if the channel-forming region of the molecule has been translocated across the membrane. Consistent with this interpretation is the ability now oftrans pepsin to destroy these reversed channels when they are closed, but not when they are open, whereascis pepsin has no effect on them in either the open or closed state. Our results indicate that voltage gating of the E1 channel involves translocation of parts of the protein across the membrane, exposing different domains to thecis andtrans solutions in the different channel states.     

Regulation of Deactivation by an Amino Terminal Domain in Human Ether-à-go-go?–related Gene Potassium Channels

Abnormalities in repolarization of the cardiac ventricular action potential can lead to life-threatening arrhythmias associated with long QT syndrome. The repolarization process depends upon the gating properties of potassium channels encoded by the human ether-à-go-go–related gene (HERG), especially those governing the rate of recovery from inactivation and the rate of deactivation. Previous studies have demonstrated that deletion of the NH₂ terminus increases the deactivation rate, but the mechanism by which the NH₂ terminus regulates deactivation in wild-type channels has not been elucidated. We tested the hypothesis that the HERG NH₂ terminus slows deactivation by a mechanism similar to N-type inactivation in Shaker channels, where it binds to the internal mouth of the pore and prevents channel closure. We found that the regulation of deactivation by the HERG NH₂ terminus bears similarity to Shaker N-type inactivation in three respects: (a) deletion of the NH₂ terminus slows C-type inactivation; (b) the action of the NH₂ terminus is sensitive to elevated concentrations of external K⁺, as if its binding along the permeation pathway is disrupted by K⁺ influx; and (c) N-ethylmaleimide, covalently linked to an aphenotypic cysteine introduced within the S4–S5 linker, mimics the N deletion phenotype, as if the binding of the NH₂ terminus to its receptor site were hindered. In contrast to N-type inactivation in Shaker, however, there was no indication that the NH₂ terminus blocks the HERG pore. In addition, we discovered that separate domains within the NH₂ terminus mediate the slowing of deactivation and the promotion of C-type inactivation. These results suggest that the NH₂ terminus stabilizes the open state and, by a separate mechanism, promotes C-type inactivation.     

N-terminal insertion of alamethicin in channel formation studied using its covalent dimer N-terminally linked by disulfide bond

Alamethicin is supposed to form helix-bundle-type channels by inserting the N terminus into bilayer lipid membranes under sufficient voltages. The N-terminal insertion has been studied with an alamethicin dimer (di-alm) N-terminally linked by a disulfide bond and by the asymmetric addition of dithiothreitol (DTT) and tetrathionate (TT) to the membrane. When di-alm was added to the cis-side membrane, it forms long-lasting channels with the lifetime τ of about 100 ms at cis-positive voltages. The lifetime was reduced to a few milliseconds by addition of DTT to the cis-side membrane, indicating that most of the channels were formed by the monomers (alm-SH) that resulted from the cleavage of the disulfide bond in di-alm. The succeeding addition of TT to the trans-side produced channels of τ=10-20 ms besides the channels of alm-SH. The results suggested that TT reacted with the N-terminal thiol group of alm-SH located at the trans-side of the membrane to alter the lifetime. The N-terminal insertion of alamethicin helices by voltage activation, therefore, was confirmed.     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an a-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two -helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 m KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Sizing the protein translocation pathway of colicin Ia channels

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include /=90 mV, even a 26-A stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is >/=26 A in diameter, or can stretch to this value. This is large enough for an alpha-helical hairpin to fit through.     

Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61α. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.     

Topology of the Shaker Potassium Channel Probed with Hydrophilic Epitope Insertions

The structure of the Shaker potassium channel has been modeled as passing through the cellular membrane eight times with both the NH₂ and COOH termini on the cytoplasmic side (Durrell, S.R., and H.R. Guy. 1992. Biophys. J. 62:238–250). To test the validity of this model, we have inserted an epitope consisting of eight hydrophilic amino acids (DYKDDDDK) in predicted extracellular and intracellular loops throughout the channel. The channels containing the synthetic epitope were expressed in Xenopus oocytes, and function was examined by two-electrode voltage clamping. All of the mutants containing insertions in putative extracellular regions and the NH₂ and COOH termini expressed functional channels, and most of their electrophysiological properties were similar to those of the wild-type channel. Immunofluorescent staining with a monoclonal antibody against the epitope was used to determine the membrane localization of the insert in the channels. The data confirm and constrain the model for the transmembrane topology of the voltage-gated potassium channel.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 μM), ATR and CAT (5-100 μM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Multinuclear NMR study and crystal structures of complexes of the types cis- and trans-Pt(amine)2I2

Complexes of the types cis- and trans-Pt(amine)₂I₂ were studied by spectroscopic methods, especially by multinuclear NMR spectroscopy. In ¹⁹⁵Pt NMR, the cis diiodo compounds with primary amines were observed between −3342 and −3357 ppm in acetone, while the trans compounds were found between −3336 and −3372 ppm. For the secondary amines, the chemical shifts were observed at lower fields. In ¹H NMR, the trans complexes were observed at higher fields than the cis compounds, while in ¹³C NMR, the reverse was observed. The ²J(¹⁹⁵Pt-¹H) and ³J(¹⁹⁵Pt-¹H) coupling constants are larger for the cis compounds (ave. 67 and 45 Hz, respectively) than for the trans isomers (ave. 59 and 38 Hz). In ¹³C NMR, the values of ²J(¹⁹⁵Pt-¹³C) and ³J(¹⁹⁵Pt-¹³C) were also found to be larger for the cis complexes (ave. 17 and 39 Hz versus 11 and 28 Hz). There seems to be a slight dependence of the pK_a values of the protonated amines or the proton affinity in the gas phase with the δ(Pt) chemical shifts. The crystal structures of eight diiodo complexes were determined. These compounds are cis-Pt(CH₃NH₂)₂I₂, cis-Pt(n-C₄H₉NH₂)₂I₂, cis-Pt(Et₂NH)₂I₂, trans-Pt(n-C₃H₇NH₂)₂I₂, trans-Pt(iso-C₃H₇NH₂)₂I₂, trans-Pt(n-C₄H₉NH₂)₂I₂, trans-Pt(t-C₄H₉NH₂)₂I₂ and trans-Pt(Me₂NH)₂I₂. The Pt-N bond distances located in trans position to the iodo ligands were compared to those located in trans position to the amines. The Pt-N bond in cis-Pt(Et₂NH)₂I₂ are much longer than the others, probably caused by the steric hindrance of the two very bulky ligands located in cis positions.     

The interactions of cis- and trans-diammineplatinum compounds with 5′-guanosine monophosphate and 5′-deoxyguanosine monophosphate. A proton nmr investigation

The interactions of cis- and trans-diammineplatinum compounds with 5′-GMP and 5′-dGMP in dilute aqueous solution at neutral pH were investigated by ¹H nmr. In addition to the 1:2 Pt nucleotide complexes cis- and trans-Pt(NH₃)₂(GMP)₂, it was possible to study the formation of the 1:1 Pt-nucleotide complexes with either one coordinated water or chloride ion. At 5°C GMP reacts with a stoichiometric amount of cis-diaquodiammine-platinum to yield cis-Pt(NH₃)₂(GMP) (H₂O) as a sole reaction product. From the present results it is concluded that such a complex may play an important role as the initial reaction product between antitumor compounds like cis-Pt(NH₃)₂Cl₂ and guanine in DNA in living organisms. The coupling constant ³J(H(1′)-H(2′)) of the H(1′) sugar proton in cis-Pt(NH₃)₂(GMP)₂ is temperature dependent, indicating a conformational change in the sugar moiety.     

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA₆₃)₇, in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA₆₃)₇ incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF_N (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF_N adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF_N in the normal direction but also can, at least partially, translocate LF_N in the opposite direction.     

Polymer Partitioning and Ion Selectivity Suggest Asymmetrical Shape for the Membrane Pore Formed by Epsilon Toxin

Using poly-(ethylene glycol)s of different molecular weights, we probe the channels formed in planar lipid bilayers by epsilon toxin secreted by the anaerobic bacterium Clostridium perfringens. We find that the pore is highly asymmetric. The cutoff size of polymers entering the pore through its opening from the cis side, the side of toxin addition, is ∼500 Da, whereas the cutoff size for the polymers entering from the trans side is ∼2300 Da. Comparing these characteristic molecular weights with those reported earlier for OmpF porin and the α-Hemolysin channel, we estimate the radii of cis and trans openings as 0.4 nm and 1.0 nm, respectively. The simplest geometry corresponding to these findings is that of a truncated cone. The asymmetry of the pore is also confirmed by measurements of the reversal potential at oppositely directed salt gradients. The moderate anionic selectivity of the channel is salted-out more efficiently when the salt concentration is higher at the trans side of the pore.     

Multiple Determinants Direct the Orientation of Signal–Anchor Proteins: The Topogenic Role of the Hydrophobic Signal Domain

The orientation of signal–anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH₂-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7–25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH₂ terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH₂-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.     

Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.