Converting a transmembrane receptor to a soluble receptor: Recognition domain to effector domain signaling after excision of the transmembrane domain

Abstract

The bacterial aspartate receptor was reconstructed to eliminate the transmembrane domain, thus connecting the recognition domain directly to the effector domain. The resulting soluble receptor folded correctly and was no longer an integral membrane protein. Upon aspartate binding, this soluble receptor was stabilized to a similar extent as that of the native receptor. Of interest, this soluble receptor retained the ability to signal from the recognition to the effector domain. This result defines more clearly the role of the membrane and transmembrane domains in signal transduction and suggests that some ligand-induced motions in receptor proteins do not require the membrane or transmembrane domain for information transmission.

Transmembrane receptors are ubiquitous in biological systems and are used to transmit information from the outside to the inside of a cell. Non-ion channel receptors consist of an exterior stimulus recognition domain, an interior effector domain (for the output response), and connecting transmembrane (TM) domains. Of these, the bacterial aspartate receptor falls into a one- to two-TM domain per receptor class that includes the insulin receptor, the epidermal growth factor receptor, and the cytokine receptors. The aspartate receptor and its homologues allow bacteria to migrate efficiently up gradients of nutrients and down gradients of toxic substances. Upon detecting its stimulus, aspartate, at the periplasmic domain, the receptor generates two responses in the cytoplasmic domain: a change in methylation of the receptor (1, 2) and alteration of a kinase cascade (3, 4).

The mechanism of TM signaling in receptors has not been determined. The one- to two-TM domain family of receptors appears to be functional as oligomers (5, 6); however, oligomerization may be only a requisite for signaling, and not the mechanism of signaling, because both the insulin and the aspartate receptors are always dimeric (7–10). Additionally, receptor chimeras between the always-dimeric insulin receptor and the epidermal growth factor receptor retain function (11), suggesting that there are additional mechanisms for signal transduction besides oligomerization. Additionally, many functionally receptor chimeras have been described, including one between the aspartate receptor and the insulin receptor (12), suggesting, furthermore, that mechanisms of signal transduction between different receptors are very similar. For the aspartate receptor, it has been proposed that ligand binding initiates a piston-type motion of TM domain II and the helices attached to it (13, 14); this has been supported by biochemical evidence (15–18). To clarify further the types of mechanisms that are possible for TM signaling, we designed a receptor lacking the TM domains and determined whether it is able to signal from recognition to effector domains.

MATERIALS AND METHODS

Plasmids and Strains.

The gene encoding the soluble receptor was constructed from the wild-type tar_S gene encoded by plasmid pEMBLtar_S (19) using a series of PCRs. The first reaction amplified the region 5′ to the sequence encoding the first TM domain, the second reaction amplified the region that was encoded between the two TM domains, and the third reaction amplified the region encoded 3′ to the second TM domain. Each of these reactions was carried out using primers with restriction sites at the end, such that digestion with the appropriate enzyme and ligation would create products that coded for a tar_S gene that was in-frame but had none of the original DNA encoding the TM domains. The regions deleted coded for amino acids 7–30 and amino acids 189–212 and were determined based on hydropathy analysis (20); the first TM domain was replaced by two amino acids, serine and arginine, and the second TM domain was not replaced by any amino acids. This tarSΔTM product was then ligated into the parent plasmid to create pOK86. The region that had been amplified was sequenced completely to verify that there were no additional changes. Escherichia coli strain HCB721 (21) was used for overproduction of the soluble receptor for protein purification. Swarm assays were performed as described (22) using E. coli strain RP437 (23).

Protein Purification.

The soluble receptor was purified as described for the wild-type aspartate receptor (24), with the following modifications. HCB721 pOK86 cells were resuspended in 100 mM sodium phosphate (pH 7.0), 10% (wt/vol) glycerol, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF), lysed by sonication, and centrifuged at 10,000 × g, 4°C, for 12 min. The resulting soft pellet contained the soluble receptor. This pellet was then resuspended, using both vortexing and homogenization, into 4 volumes of 20 mM sodium phosphate (pH 7.0), 2 M KCl, 10% (wt/vol) glycerol, 5 mM EDTA, and 1 mM PMSF and recentrifuged as above. This was repeated two times. Finally, the pellet was washed once with 50 mM Tris⋅HCl (pH 7.5) at 4°C, 10% (wt/vol) glycerol, 5 mM EDTA, and 1 mM PMSF and resuspended into this to give a final volume of 1 g of cell paste/1.5 ml. This was stored at −20°C for up to 9 months.

To complete the purification, a 15-ml aliquot of the above was thawed, n-octyl β-d-glucoside (OG) (Sigma) was added to a final concentration of 1.5% (wt/vol), and this was gently shaken at 4°C for 30 min. Insoluble material was removed by centrifugation at 370,000 × g in a TLA 100.3 rotor (Beckman). The supernatant was applied to a 20-ml ω-aminooctylagarose column (Sigma), preequilibrated with 50 mM Tris (pH 7.5) at 4°C, 5 mM EDTA, 10% (wt/vol) glycerol, 1% OG, and 1 mM PMSF and eluted with a linear NaCl gradient from 0 to 400 mM in the same buffer. Soluble receptor-containing fractions were pooled and dialyzed against 21 volumes of the same buffer without NaCl. This was then concentrated to ≈2 mg/ml and stored at −80°C until needed.

Aspartate Binding Assay.

The aspartate affinity for the soluble receptor was determined using a modification of the competition centrifugation assay (24), except that 30,000-Da cutoff filter units were used, and the amount of aspartate remaining in solution was determined by counting triplicate 4-μl aliquots, mixed with 100 μl of H₂O in 3 ml of scintillation fluid. Aspartate binding data were analyzed by Scatchard plots (25) and the Hill equation (26) as described by Kolodziej and coworkers (27).

CD Spectroscopy.

CD spectra were collected using a Bruker (Billerica, MA) spectrophotometer that had a thermostatically controlled cell holder. For temperature melts, ellipticity data at 222 nm were obtained using a 1-mm path length suprasil quartz cuvette (Starna, Atascadero, CA), from 4° to 90°C in 2°C intervals. Equilibration times of 2.5–3 min and averaging times of 30 s were used. For wavelength scans, ellipticity data were obtained from 300 to 190 nm at 4°C using a 5-s averaging time and no equilibration time. Protein samples were prepared for CD by dialyzing against 10 mM sodium phosphate buffer (pH 7.0; soluble receptor) or 10 mM sodium phosphate buffer (pH 7.0) plus 1% OG (wild-type aspartate receptor). A scan of the buffer that the protein was in, ±aspartate, was used as a blank and subtracted from all data. The final protein concentration was between 0.13 and 0.36 mg/ml and was determined by Bradford assay (28) using BSA as a standard.

Methylation Assays.

Methylation assays were performed essentially as described (29, 30). S-Adenosyl[³H]methionine (Amersham) was used at a final concentration of 3.35 μM, glycerol was added to the assay to a final concentration of 40% (wt/vol), and aspartate was used at 0.1 mM (aspartate concentrations from 0.1 to 3 mM gave similar results). The soluble receptor was prepared by dialyzing purified protein against 250 volumes of 50 mM Tris (pH 7.5) at 4°C, 5 mM EDTA, 10% (wt/vol) glycerol, and 1 mM PMSF at 4°C in 10–12,000-Da cutoff Slidealyzers (Pierce) to remove the OG. After dialysis, the protein concentration was determined by Bradford assay (28) with BSA as a standard, and the protein was diluted and used at 0.4 mg/ml.

RESULTS

The Soluble Receptor Is Functionally Similar to the Native Receptor.

To produce an aspartate receptor that has no TM domains, deletions of the regions of the tar_S gene encoding these domains were made using the PCR (Fig. 1). TM domain I (amino acids 7–30) was deleted and replaced with two amino acids, serine and arginine, and TM domain II (amino acids 189–212) was deleted and replaced with no amino acids. This new protein is ≈5 kDa smaller than the native aspartate receptor and is retained in the cytoplasm, and its identity was confirmed by sequencing the modified gene and by reaction of the protein with anti-aspartate receptor antibodies.

Figure 1.

The soluble aspartate receptor is missing both TM domains. The ligand binding/recognition domains are shown as thick lines, the TM domains are shown as zigzag lines, and the cytoplasmic/effector domains are shown as thin lines.

Overexpression of the soluble receptor results in a dominant negative phenotype that is almost identical to that produced by the native receptor: the swarm rate of wild-type E. coli is decreased when the receptor is overproduced (Table 1) (20). This suggests that the cytoplasmic domain of the soluble receptor is able to fold in vivo into a structure that is similar to that of the native aspartate receptor and interact with downstream chemotaxis proteins. To characterize the ligand-binding properties of the soluble receptor, the dissociation constant for aspartate, as well as the cooperative behavior of the receptor, was determined using the modified competition centrifugation assay (Fig. 2). Both the binding constant for aspartate, 1.65 ± 0.2 μM, and the negative cooperativity (Hill coefficient of 0.66) were essentially the same as for the native receptor (24). Thus, the ligand-binding domain of the soluble receptor appears to be similar to that of the full length receptor both in its ability to bind aspartate and in exhibiting the intersubunit interactions that are necessary for negative cooperativity.

Figure 2.

The soluble receptor binds aspartate with negative cooperativity and a dissociation constant (K_d) of 1.65 ± 0.2 μM. Binding affinity of the soluble receptor was determined using a modified equilibrium binding assay with l-[³H]aspartate. Data from this assay were plotted in the form of Scatchard.

Aspartate-Binding Stabilizes the Soluble and Native Receptors.

To test the relative stabilities of the receptors, the effect of temperature on the structure of these proteins was studied using the CD spectroscopy signal at 222 nm (Fig. 3). As the temperature increases, the protein unfolds, and there is a loss of structure (31); this method can be used to measure differences in stability of two proteins. Fig. 3a shows the melting curve for the native aspartate receptor in the presence and absence of aspartate; the melting temperature, T_m, is 38°C in the absence of aspartate and 62°C in the presence of aspartate. Fig. 3b shows the melting curve of the soluble receptor in the presence and absence of aspartate; the T_m is 40°C in the absence and 56°C in the presence of aspartate. Thus, although the soluble receptor is somewhat less stable than the native receptor, aspartate binding induces similar structural changes that stabilize the native and soluble receptors to comparable extents.

Figure 3.

Thermal denaturation of native and soluble aspartate receptors. CD signals of the native (a) and soluble (b) receptors were obtained at 222 nm, from 4° to 90°C, in the absence (open symbols) and presence (closed symbols) of 4.5 mM aspartate. (Inset, b) The full CD spectrum of the soluble receptor from 300 to 185 nm in the presence and absence of aspartate.

The Soluble Receptor Can Signal from Recognition to Effector Domain.

In the native receptor, aspartate binding causes an increase in the rate of methylation of the effector domain by the CheR methyltransferase. To determine whether the soluble receptor would transmit signals between these two domains, we monitored the change in methylation rate upon the binding of aspartate. As shown in Fig. 4, the soluble receptor is methylated, and the rate of this methylation increases 1.25-fold upon addition of aspartate. This behavior is similar to that of the native receptor; however, the magnitude of the aspartate-induced change is smaller; the increase in the native receptor is usually 1.5–2-fold (27, 29). Additionally, the rate of methylation of the soluble receptor is ≈5-fold diminished compared with the native membrane-bound receptor; however, the rate of methylation of native receptor, solubilized in OG and then dialyzed to remove the OG (prepared the same way as the soluble receptor), was essentially the same as background and did not show an aspartate-induced rate increase (data not shown). Thus, this indicates that the soluble receptor, which is missing the TM domains and is not localized to the membrane, is able to transmit information from recognition to effector domain.

Figure 4.

The rate of soluble receptor methylation increases upon addition of aspartate. Methylation assays were performed as described in Materials and Methods. No-receptor reactions were done with protein prepared in the same way as the soluble receptor, except that E. coli HCB721 contained only plasmid pEMBL18 (no receptor encoding insert) and were used at 3-fold higher protein concentrations. The graph shows representative data from one experiment. This experiment was repeated 44 times; 34 of these showed an aspartate-induced rate increase that averaged 1.25-fold.

DISCUSSION

To understand further the types of ligand-induced motions in receptor proteins, we created a soluble aspartate receptor in which the recognition site has been grafted onto the effector domain. This receptor binds aspartate and is able to transmit ligand-induced conformational changes from the recognition domain to the effector domain. These results suggest that some ligand-generated motions in receptor proteins do not require membrane, or the presence of TM domains, for the transmission of information from recognition to effector domains.

In the aspartate receptor, several ligand-induced motions have been postulated. In one, the piston-type motion, ligand binding generates movement perpendicular to the membrane, such that the recognition domain and/or the effector domain are moved closer or further from the membrane (13). This model has been extensively supported by both biophysical and crystallographic evidence (14, 16–18). It is also apparent, however, that aspartate binding initiates a closer association of the two subunits (32). This has been supported by experimental evidence from disulfide cross-linking (15, 19, 33), lack of interchange between subunits (7), and the determination that the aspartate binding site is at the interface between the two subunits (32). Additional evidence, using leucine zipper fusion proteins, random mutagenesis of the TM domains, and analysis of the crystal structure, has suggested that ligand binding might induce a rotation between the two aspartate receptor subunits (32, 34, 35). Because the soluble receptor is not membrane-localized, ligand-induced motions would not be able to push or pull one domain relative to the membrane. Thus, in the soluble receptor, the major ligand-generated motions that remain would involve the closer association or rotation of the two subunits relative to each other. This is a surprising result given that previous evidence suggested that ligand-induced motions required only one subunit (36–38). The most consistent explanation, however, is that the ligand-binding in the native membrane-bound receptor induces conformational changes that are comprised of several motions. The diminished response of methylation in the absence of TM domains and membrane suggest that some features, e.g., the vertical movement relative to the membrane, play less of a role in the soluble receptor whereas others, e.g., the horizontal movements, play a larger role in the soluble receptor.

Previous work has been somewhat inconclusive in regard to the role of TM domains in receptor molecules: Whole domains can be substituted in some cases (39, 40) whereas some single amino acid mutations can radically alter protein function (41–43). In addition, several receptor deletions encompassing the TM domain that retain partial in vivo function have been described (44–46).

The fact that a designed soluble receptor can signal has valuable practical features. Such soluble receptors, by virtue of their solubility, will be much more amenable to biophysical and biochemical techniques and thus should prove to be extremely helpful in understanding “TM-signaling” mechanisms. Thus for both theoretical and practical reasons, soluble receptors with excised TM domains appear to be a valuable tool for the future.

Table 1.

Overexpression of wild-type and soluble aspartate receptors is dominant-negative

RP437 plus	Swarm rate, mm/h
	Tryptone	Min.	Min. + Asp	Min. + Ser
Plasmid only	4.04 ± 0.89	0.65 ± 0.19	1.69 ± 0.5	1.77 ± 0.17
Native aspartate receptor	1.63 ± 0.58	0.55 ± 0.17	0.77 ± 0.17	0.70 ± 0.17
Soluble aspartate receptor	1.33 ± 0.42	0.61 ± 0.09	0.59 ± 0.07	0.58 ± 0.12

The swarm rate was determined on plates containing 0.3% agar of E. coli RP437 overexpressing the wild-type receptor (pEMBLTarS), the soluble receptor (pOK86), or no receptor (pEMBL18). Swarm rates are given as the rate of colony expansion, in mm/h, and represent the average of seven assays. Min., minimal glycerol media; Min. + Asp, minimal media with 100 mM aspartate; Min. + Ser, minimal media with 100 mM serine.

ABBREVIATIONS

TM

transmembrane

PMSF

phenylmethylsulfonyl fluoride

OG

n-octyl β-d-glucoside