Department of Molecular and Cell Biology, 329 Stanley Hall, University of California, Berkeley, CA 94720, USA
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Abstract |
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Keywords: chemotaxis/helixhelix interactions/signal transduction/transmembrane sequence
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Introduction |
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One example where this sort of 'biased-random' mutagenesis would be important is transmembrane proteins. The Escherichia coli aspartate receptor is a model system for the study of transmembrane protein structure and function. It contains a periplasmic ligand binding domain, two transmembrane sequences and a cytoplasmic signaling domain per subunit (Mowbray et al., 1985; Falke and Koshland, 1987
), as shown in Figure 1
. It is a dimer in both the presence and absence of aspartate, thus a complete receptor has four transmembrane sequences (
). Cross-linking experiments indicated that each transmembrane sequence probably forms a helix (Lynch and Koshland, 1991
; Pakula and Simon, 1992
).
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Materials and methods |
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[-32P]ATP was from Amersham. GeneClean kits were from Bio101 (La Jolla, California). T4 nucleotide kinase and T4 DNA ligase were from Boehringer Mannheim. Sequenase kits were from US Biochemical Corporation. Mutagene kits were from BioRad. Aspartate, ampicillin and other chemicals were from Sigma.
Strains
Escherichia coli strain CJ236 (dut ung) was purchased from Biorad, E.coli strain DH5 was purchased from Bethesda Research Laboratories. Escherichia coli strains RP5838 (tar tap tsr) and RP4372 (tar tsr) were gifts from J.S.Parkinson. Epicurean coli strain XL1-Blue was purchased from Stratagene.
Construction of TMIR mutant
Plasmid pApaI (constructed by Gideon Bollag, Ph.D. thesis) encodes a mutant E.coli tar gene with a deletion of the second transmembrane sequence and an engineered ApaI restriction site in its place. Synthetic oligonucleotides for the coding and noncoding strands of the insulin receptor transmembrane sequence (GB1 and GB2 in Table I
) were annealed to each other and ligated into the p
ApaI vector. Ligation mixes were used to transform XL1-Blue competent cells. The sequence of the second transmembrane region was confirmed by double-stranded sequencing.
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The sequence for the 24R, 7RI and 7RII oligonucleotides are given in Table I. The 24R oligonucleotide binds to 20 nucleotides 5' to the second transmembrane coding sequence and 20 nucleotides 3' to the second transmembrane coding sequence. It replaces the second transmembrane sequence (residues 189212) with 24 'hydrophobic-biased' random amino acid residues. The 7RI and 7RII oligonucleotides replace residues 199205 with 'hydrophobic-biased' random residues. The ratios of the nucleotides for each position in the 'hydrophobic-biased' codons were chosen so that the majority of the codons would encode hydrophobic or polar residues and very few would be nonsense codons or encode charged residues, as described in the text. The 24R mutagenic oligonucleotides were used in site-directed mutagenesis of pMK650, and the 7RI and 7RII oligonucleotides were used in site-directed mutagenesis of pCJM7 by the method of Kunkel (1985). pCJM7 is a derivative of pMK650 in which the codons encoding residues 199205 were deleted.
Behavioral assay
Fresh transformants of strains that lack a chromosomally encoded aspartate receptor (RP5838 or RP4372) were used to inoculate minimal + aspartate soft agar plates, referred to as 'swarm' plates. In each experiment, fresh transformants in the same strain were used for positive controls (which expressed the wild type receptor from plasmid pMK650) and negative controls (which contained vector alone, pEMBL19, and expressed no aspartate receptor). Swarm plates contained 100 µM aspartate, 0.3% agar, 100 µg/ml ampicillin and 500 mg/l of each of the following: L-histidine, L-methionine, L-leucine, L-threonine and thiamine. Swarm plates were allowed to solidify at least 4 h, usually overnight. Plates were inoculated with a drop of overnight culture using a sterile wooden stick. Swarm plates were incubated at 30°C. Swarm diameters were measured five or six times over an 810 h period starting approximately 2024 h after inoculation. Swarm rates were defined as the slope of a linear curve fit of the swarm diameters.
Expression of mutant receptors in RP5838
300 ml minimal 100 µg/ml ampicillin cultures were inoculated with a 5 ml LB 200 µg/ml ampicillin overnight culture grown at 30°C. The cultures were grown for 7 h with shaking at 30°C. One ml of the culture was used for the preparation of whole cell extracts. The remaining culture was used to make membrane preparations as described below. Whole cell extracts were prepared as follows: 1 ml samples were centrifuged, and the cell pellet was resuspended in SDS sample buffer so that the final concentration of total cell protein was the same in each extract. The extracts were subjected to three rounds of freezing and boiling to solubilize the membrane proteins. Samples were run on two identical 7.5% SDSpolyacrylamide gels by the method of Laemmli (1970), and the proteins were transferred to nitrocellulose. The blot was probed with a polyclonal anti-tarE antibody (#9207 prepared by H.-P. Biemann), and the overexpressed band was confirmed as the aspartate receptor.
Test for insertion of mutant receptors into the cell membrane
The remaining culture from the receptor expression experiment was subjected to low speed centrifugation, and the cell pellet was frozen in liquid nitrogen and stored at 80°C. The cell pellet (approximately 0.5 g) was used to prepare membrane samples as described previously (11) except for the following changes: buffer A contained 100 mM sodium phosphate, pH 7.0, 10% glycerol, 5 mM EDTA, 5 mM phenanthroline and 1 mM PMSF. Buffer B contained 20 mM sodium phosphate, pH 7.0, 2 M KCl, 10% glycerol, 5 mM EDTA, 1 mM phenanthroline and 0.5 mM PMSF. Membranes were sedimented by centrifugation at 100 000 r.p.m. in a Beckman type TLA 100.3 rotor at 4°C. After the first high speed centrifugation step, a sample of the supernatant was saved as the 'cytoplasmic fraction'. After the second wash in buffer B, the membrane pellet was resuspended in 300 µl final buffer (10 mM TrisHCl, pH 7.5, 10% glycerol, 5 mM phenanthroline and 1 mM PMSF). Samples of the cytoplasmic fraction and the membrane preparation from each culture were separated on 7.5% polyacrylamide gels, and the proteins were transferred to nitrocellulose. Sample size was chosen so that the density of the band would be proportional to the amount of receptor loaded on the gel (i.e. the nitrocellulose was not saturated at these levels of protein). The blots were probed with a polyclonal anti-tarE antibody as described above.
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Results |
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To determine whether or not the aspartate receptor could tolerate the substitution of a transmembrane helix from an unrelated receptor in place of one of its own transmembrane helices, a mutant receptor, TMIR, was constructed in which the second transmembrane helix of the aspartate receptor was replaced with the transmembrane helix of the insulin receptor. The TMIR mutant was constructed by ligation of a synthetic oligonucleotide cassette that encodes the insulin receptor transmembrane sequence into the ApaI site of plasmid pApaI.
The transmembrane sequences of TMIR, the insulin receptor and the aspartate receptor are shown in Table II. Because of the location of the restriction site, the transmembrane sequence in TMIR differs from the transmembrane sequence of the insulin receptor at the N-terminal residue, which is changed from an isoleucine to an alanine, and the two C-terminal residues, which are changed from a phenylalanine to a glycine and from a leucine to an alanine.
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A strain that lacks a chromosomally encoded wild type aspartate receptor, RP4372, was transformed with the TMIR plasmid. Control transformants were RP4372(pMK650), which encodes the wild type aspartate receptor, and RP4372(pEMBL19), which does not encode an aspartate receptor. Transformants were used to inoculate aspartate swarm plates. Cells expressing the wild type receptor had a swarm rate of over 0.5 mm/h (Figure 2A). The negative control had a swarm rate under 0.2 mm/h. Cells expressing the TMIR mutant also had a low swarm rate in the presence of aspartate and formed small, dense colonies like that formed by the RP4372(pEMBL19) transformants. These results indicate that the TMIR receptor does not respond to aspartate and so is not effectively functional in chemotaxis.
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'Hydrophobic-biased' random mutagenesis
In order to study the effects of changing the sequence of the entire transmembrane helix independent of a change in length, we used a variation of site-directed mutagenesis, which we call 'hydrophobic-biased' random mutagenesis, to replace the second transmembrane sequence, residues 189212, with random hydrophobic and polar residues. The ends of the mutagenic oligonucleotide contained sequences complementary to the sequences 5' and 3' to the aspartate receptor second transmembrane coding sequence and the 24 codons in the middle of the sequence were random. The ratios of the four nucleotides at each position in the randomized codons were calculated as described below so that the probability that each random codon encoded a hydrophobic or polar residue was high but the probability that it encoded a charged residue or was a nonsense codon was low.
As shown in Table III, we divided the 64 codons into two categories: those that encode hydrophobic or polar residues (F, Y, W, G, A, V, I, L, M, C, T, S, N, Q and P) and those that encode charged residues or are nonsense codons (H, K, D, E, R and nonsense). A random codon, XYZ, with equal amounts of A, T, C or G in each position X, Y and Z, would encode a residue in the first group 67.2% of the time and encode a residue in the second group 32.8% of the time (as indicated in Table III
, column labeled A = T = C = G). This means that a random sequence of 24 amino acids would include an average of seven charged residues and would be unlikely to serve as a transmembrane sequence. In order to increase the probability that the codons encode residues in the hydrophobic and polar group, we altered the ratios of A, T, C and G for each position in the random codon, X, Y and Z.
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After these initial adjustments, we performed several rounds of optimizing the nucleotide ratios as follows: (i) calculate the probability for the random codon to encode each residue and the total probabilities that the codon encodes any residue from the hydrophobic/polar set or the charged/nonsense set; (ii) adjust the percentage of each nucleotide in each position, X, Y or Z. Then, calculate the new probabilities for individual residues and for the two groups of residues. If the adjustment increased the probability that the codon encoded a polar or hydrophobic residue, it was kept, and if it did not, it was discarded. After eight rounds of adjustments and calculations, the amount of improvement decreased, and we chose the best ratios of A, T, G and C for each position, X, Y and Z, for the random regions of our mutagenic oligonucleotides (Table I). For the 24R and 7RI oligonucleotides, the probability that each random codon encodes alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, tyrosine, serine, threonine, glycine, cysteine, proline, asparagine or glutamine is 94.9%, but the probability of each codon encoding a nonsense codon, arginine, glutamate, lysine, histidine or aspartate is 5.1%.
After obtaining the results from the experiments described below with mutant receptors made using the 24R and 7RI oligonucleotides, we designed an additional oligonucleotide, 7RII, where there was a more equal probability of the random codon encoding each amino acid. For example, the probability of the random codon in the 24R and 7RI oligonucleotides encoding N, Q or Y was 0%. In addition, the probability that the codon would encode V or L (25.5 and 19.1%, respectively) was much greater than for the other hydrophobic or polar residues (Table III). Also, in some experiments, it might be desirable to allow the codon to encode some of the charged residues, too. We repeated the calculations to determine new ratios of A, T, C and G for each position X, Y and Z. For these ratios, we started with a small amount of A in the 'Y' position and then repeated the rounds of calculating the individual probabilities and making adjustments in the nucleotide ratios. For the 7RII oligonucleotide, the probability of each random codon encoding alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, tyrosine, serine, threonine, glycine, cysteine, proline, asparagine or glutamine was 94.5%, and the probability of each codon encoding a nonsense codon, arginine, glutamate, lysine, histidine or aspartate was 5.5%. The probabilities that the random codons encode each hydrophobic and polar residue are more equal, with a decrease in the probabilities for V and L in favor of some of the other hydrophobic residues that previously had had very low probabilities (F, Y, I, C, N, Q and S) (Table III
). [The probabilities for a few of the other hydrophobic and polar amino acids were also unavoidably slightly decreased (W, G, A, M and T).] The probabilities that the codon encodes an asparagine, glutamine or tyrosine were increased.
Receptors with 24 'hydrophobic-biased' random residues
The 24R oligonucleotide was used in 'hydrophobic-biased' random mutagenesis to construct four plasmids encoding mutant receptors in which the entire second transmembrane sequence was replaced with 'hydrophobic-biased' random residues. The sequences of these mutant receptors are shown in Table II. Mutants 24R1, 24R3 and 24R5 had only two, four and two residues the same as the wild type sequence, respectively. Mutant 24R6 had 14 residues the same as the wild type sequence, which was probably due to the increased probability of an oligonucleotide binding to the single-stranded vector when it has more nucleotides complementary to the vector.
The ability of each of the four mutant receptors to respond to aspartate was determined in swarm plate assays in a strain that lacks a chromosomally encoded aspartate receptor, RP5838. The swarm rates from these experiments are shown in Figure 2B. Transformants that expressed receptors 24R1, 24R3, 24R5 or 24R6 had lower swarm rates than cells expressing the wild type receptor, but the appearance of the colonies was larger and more diffuse than what is seen with vector alone. The appearance of these colonies suggests that these transformants may have a small ability to spread out towards a higher concentration of aspartate. However, their response is far less than that of cells expressing the wild type protein and too small to be measured by the swarm plate assay, so each of these four receptors was categorized as having a 'borderline' response to aspartate (Table II
).
Receptors with seven 'hydrophobic-biased' random residues
It is possible that the receptors with 24 amino acid substitutions only exhibited a slight response to aspartate because of the number, nature or location of the substitutions within the second transmembrane sequence. In a second set of mutant receptors, the residues from position 199 to 205 in the transmembrane sequence, only seven residues, were replaced with 'hydrophobic-biased' random residues. These residues are likely to form only two turns of a transmembrane helix. The mutant receptors contain 10 wild type amino acid residues at the N-terminal end of the transmembrane sequence and seven wild type amino acid residues at the C-terminal end of the transmembrane sequence, so the substituted positions were not near the ligand binding or signaling domains of the receptor. These mutant receptors were constructed by site-directed mutagenesis using the 7RI oligonucleotide or the 7RII oligonucleotide. Thirteen plasmids encoding mutant receptors were isolated, and the sequences of the second transmembrane helices are given in Table II.
The response of each mutant receptor to aspartate was characterized using the swarm plate assay with strain RP5838. The swarm rates for these receptors are shown in Figure 2C and D. Eight of the mutant receptors enabled the cells to respond to aspartate as well as the wild type receptor and were classified as 'swarming' (Table II
). The other five mutant receptors did not enable the cells to respond to aspartate and were classified as 'nonswarming'.
Tolerated substitutions
The sequences of the receptors in the 'swarming' class indicate amino acid substitutions that are tolerated in positions 199205. These substitutions include several substitutions of polar residues in place of hydrophobic residues, such as serine in place of leucine at position 203 in mutants 7RI-1 and 7RI-2 and threonine in place of leucine at position 205 of 7RI-1 and position 199 in 7RII-8. Other acceptable substitutions include the substitution of bulky residues in place of smaller residues, such as tryptophan in place of leucine at position 199 in mutants 7RI-4 and 7RII-72, tyrosine in place of valine at position 200 in mutant 7RII-8 and phenylalanine in place of leucine in position 203 in mutant 7RII-8. Smaller residues in place of larger residues are also tolerated, such as alanine in place of valine at position 200 of mutants 7RI-1 and 7RI-2 and valine in place of leucine at position 199 of mutant 7RI-2 and position 205 of mutant 7RII-72.
Each of the seven positions (199205) tolerates multiple kinds of substitutions. The tolerated mutations found for each position are summarized in Figure 3. For example, the leucine at position 199 can be replaced by valine, tryptophan, methionine, threonine or glutamine and the receptor retains its ability to respond to aspartate. The valine at position 200 can be replaced by alanine, tryptophan, tyrosine, glycine or isoleucine and the receptor responds to aspartate. The results from these mutants do not indicate whether all combinations of these substitutions are tolerated. They also do not indicate that these substitutions include all possible tolerated substitutions at these positions.
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Two of the five 'nonswarming' receptors contain nonsense codons. Mutant 7RII-22 contains a nonsense codon at position 205. Mutant 7RII-27A contains a frameshift mutation which results in a nonsense codon several codons downstream. The presence of a nonsense codon in this region of the receptor would result in the synthesis of a receptor fragment that includes only the periplasmic and transmembrane domains. The fragment produced would lack the cytoplasmic signaling domain and would not be able to interact with the rest of the chemotaxis signaling pathway. These mutant receptors were not studied further.
The sequences of the other 'nonswarming' mutants suggest substitutions that are not tolerated in the transmembrane sequence, but in some cases the introduction of several changes simultaneously prevents a determination of the significant change or combination of changes. Receptors 7RII-27B and 7RII-73 contained an arginine at position 205. The residues at positions 199204 in these receptors include wild type residues and the kinds of substitutions that were found in the 'swarming' receptors. The final 'nonswarming' receptor, 7RII-96, contained only those kinds of substitutions that were found in the 'swarming' receptors.
A decrease in swarm rate of the transformants could be due to a large decrease in the amount of receptor in the cell or to incorrect targeting of the receptor to the membrane. Whole cell extracts and membrane preparations from cells expressing a 'nonswarming' receptor, 7RII-27B, 7RII-73 or 7RII-96, were compared in immunoblots with equal amounts of whole cell extracts and membrane preparations from cells expressing a 'swarming' receptor (7RII-8, 7RII-62 or 7RII-72) or the wild type receptor (pMK650) (Figure 4). The plasmid used to construct the mutant receptors, pCJ2, was constructed from pMK650, so the plasmids encoding the 'hydrophobic-biased' mutant receptors differ from pMK650 only in the region encoding the second transmembrane sequence. The wild type receptor and all six of the mutant receptors run as multiple bands with an apparent molecular weight of approximately 67 kDa. These bands are not seen in the lane containing an extract from cells that were transformed with vector alone (pEMBL19). The small differences in mobility of the wild type and mutant receptors were apparently due to differences in amino acid sequence or methylation state. A similar amount of receptor protein is seen from cells transformed with a vector encoding the wild type receptor, the 'swarming' receptors and 'nonswarming' receptor 7RII-96.
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To test for insertion of the mutant receptors into the cell membrane, membranes were isolated from cells expressing each of these six receptors. Membrane samples and cytoplasmic fractions were immunoblotted with anti-aspartate receptor antibodies (Figure 5). Like the wild type aspartate receptor, all of the mutant receptors tested localized to the membrane fraction. The results from the whole cell extracts and membrane preparations indicate the 'nonswarming' receptors 7RII-27B, 7RII-73 and 7RII-96 are expressed and inserted into the cell membranes. Receptor 7RII-96 is found at a similar level as the wild type receptor and inserted in the cell membrane, so changes in the swarm rate from this receptor must be due to changes in receptor folding or transmembrane signaling.
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Since the receptors with only seven substitutions could be clearly divided into 'swarming' and 'nonswarming' receptors, we can compare the 'success rate' of individual residues by comparing the number of times each residue is found in a 'swarming' receptor to the number of times it is found in a 'nonswarming' receptor (Table III, columns S and N). As indicated above, receptors with arginine or a nonsense codon did not support swarming. The small hydrophobic and polar residues, G, A, V, C, T and S were present 44 times, and 36 of these occurrences (81.8% of the time) were in 'swarming' receptors. The medium sized hydrophobic residues were present 21 times, and 14 of these occurrences (66.6% of the time) were in 'swarming' receptors. The aromatic residues, F, Y and W, were present nine times, and five of these occurrences were in 'swarming' receptors (55.6% of the time).
The 'least successful' residues were L (only six out of 11) and F (only one out of five). Three of the four occurrences of phenylalanine in 'nonswarming' receptors were in the same mutant receptor. In addition, F and L are found in the transmembrane sequences of the wild type E.coli and S.typhimurium aspartate receptors a total of eight and 24 times, respectively (Table III, column w.t.). From our results, it appears that the 'success rate' of a hydrophobic or polar residue is due largely to the context in which it is found. It may be affected by the total number of substitutions within the same receptor, its location within a transmembrane sequence or a combination of number and location.
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Discussion |
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The results described above demonstrate the feasibility of producing groups of mutant proteins with 'biased' random amino acid substitutions, where each random codon has carefully chosen probabilities of encoding selected amino acids. The 24R, 7RI and 7RII oligonucleotides contain random codons that were heavily biased towards encoding hydrophobic and polar residues. All three resulted in transmembrane sequences containing the expected hydrophobic amino acids (Table III).
The ratios of the nucleotides in each position of the 'hydrophobic-biased' random codons were calculated manually through several rounds of examining the genetic code, making changes in the ratios of adenine, thymidine, guanine and cytosine for each position, X, Y and Z, and then recalculating the new probabilities of the codon encoding each amino acid. In the future, similar 'biased-random' oligonucleotides can be designed more easily because, during the preparation of this manuscript, a computer program that can optimize the nucleotide ratios automatically became available on the Internet at http://www.wi.mit.edu/kim/computing.html (Wolf et al., 1999).
The use of multiple random substitutions provides a means of testing the results of many different changes with only a few dozen mutant receptors. The inter-relatedness of the three general characteristics that are found to be important (location, amino acid type and number of substitutions), would not have been observed with 24 mutant receptors each containing a single individual substitution, such as in alanine-scanning mutagenesis. For example, our results suggest that at a particular position in the helix, an alanine might have an effect and a serine might not, but at another position the opposite could be true. In addition, since the number of substitutions is also a factor, it appears that each individual substitution might have a very small effect on receptor function. Therefore, it is likely that testing hundreds of mutant receptors with individual substitutions might not clarify the importance of location and amino acid type unless a much more sensitive assay for receptor function can first be found.
Sequence specificity
The results of the swarm plate experiments with the 'biased-random' mutant aspartate receptors, along with the results from other mutant aspartate receptors, increase our understanding about the sequence specificity of transmembrane regions (Lynch et al., 1991; Jeffery et al., 1994; Maruyama et al., 1995
; Tatsuno et al., 1995). In general, substitutions that are found in mutant receptors that can still respond to aspartate include smaller to larger residues, larger to smaller residues, hydrophobic to polar residues and polar to hydrophobic residues. Two of the receptors that did not enable the cells to respond to aspartate (7RII-27B and 7RII-73) contained a charged residue, arginine, in the transmembrane sequence. Simon and co-workers demonstrated that the introduction of a charged residue into the first transmembrane sequence of the aspartate receptor also caused a decrease in receptor function (Milburn et al., 1991
). While 7RII-27B and 7RII-73 were expressed and inserted into the cell membrane, they were found at a lower level than the wild type receptor. This could be due to a higher rate of receptor degradation if the charged residue affected the folding of the receptor, or it could affect the interaction of the receptor with the membrane lipids.
Since none of the receptors in which the whole transmembrane sequence was replaced retained the full response to aspartate, but some of the receptors with only a few substitutions were able to respond to aspartate, it appears that the number or position(s) of substitutions can be a factor. If some of the substitutions caused small disruptions in protein structure or transmembrane signaling, the combination of many of these changes may decrease the level of receptor function. It is also possible that some of the residues in the transmembrane sequence can be changed to other hydrophobic residues, but not all the positions tolerate substitutions. Substitutions at certain positions in the transmembrane sequence may not be tolerated if that portion of the transmembrane sequence interacts with another part of the receptor. For example, if the residues near the ends of the transmembrane sequences interact with the N- or C-terminal domains of the receptor, a substitution near these positions may cause a change in the conformation of one or both of these domains. Receptor folding or signaling could also be affected if mutations occurred in a region that interacts with the other transmembrane sequences in the dimer. The substitutions in the 7RI and 7RII receptors were made in the middle of the transmembrane sequence because this region is likely to interact only with the other transmembrane sequences in the receptor. Some of the receptors with only a few substitutions had as strong a response to aspartate as the wild type receptor, which indicates that at positions 199205, the wild type sequence precisely as it is in nature does not appear to be required for correct folding and transmembrane signaling.
However, the receptor 7RII-96 contained only a few conservative substitutions near the center of the transmembrane helix and did not respond to aspartate. Since the amino acid side chains project from the sides of an -helix, the mutagenesis would alter the structure of the surface of the helix. Since the substituted residues are hydrophobic and the lipid bilayer is flexible, the substitutions would probably not impair function by altering the receptor's interaction with the lipid bilayer, but it is more likely that the specific side chains at some of these positions may prevent correct packing of this helix against the other transmembrane helices.
Cross-linking experiments demonstrated the two first transmembrane helices in the dimer are located next to each other, and each second transmembrane helix is located near the first transmembrane helix in the same subunit (Lynch and Koshland, 1991; Pakula and Simon, 1992
). This arrangement of helices is consistent with a transmembrane model that was constructed by extending the first and fourth helices in the four helix bundle of each subunit in the aspartate receptor N-terminal domain crystal structure (Milburn et al., 1991
). In a related mutagenesis study, it was found that even a single phenylalanine in place of the isoleucine at position 204 can impair receptor signaling (Jeffery et al., 1994
). The ability of a single phenylalanine in place of isoleucine substitution at position 204 to affect transmembrane signaling suggests that the side of the second transmembrane helix containing position 204 faces the other transmembrane helices in the dimer and the helices must be arranged very closely if a small change that makes the surface of the helix protrude a fraction of an Ångström can affect function. The results with the random mutants support the model that the first and second transmembrane helices within each subunit pack against each other. Also, transmembrane signaling by this receptor is predicted to involve a conformational change that alters the relative positions of the transmembrane helices (Chervitz and Falke et al., 1995, 1996). Changing the structure of the surface of these helices through replacing amino acid side chains may affect transmembrane signaling by interfering with their packing or movement.
The decrease in receptor signaling caused by fairly conservative substitutions is somewhat surprising due to the lack of sequence conservation within the second transmembrane sequences in the chemotaxis receptors. However, the overall relative arrangement of the transmembrane helices may be similar in these receptors, since the change of a residue in the second transmembrane sequence could be compensated for by a complementary change of a residue in the first transmembrane sequence.
Contrast with signal sequences
The sequence specificity seen in the aspartate receptor second transmembrane sequence is in contrast to the wide variation allowed in another class of hydrophobic amino acid sequences, the signal sequences for membrane insertion or transmembrane transport of polypeptide chains. Although both kinds of amino acid sequences are characterized by a series of hydrophobic residues, the signal sequences of at least a few proteins can be replaced by a large variety of hydrophobic sequences, as long as the overall hydrophobicity is maintained (von-Heijneet al., 1985; Ferenci-etal-1987" Ferenci et al., 1987; et al., 1987
; Gierasch, 1989
; Kaiser and Botstein et al., 1990; Izard and Kendall, 1994
). This means that a wide variety of sequences can be used by the signal recognition particle.
The sequence specificity of transmembrane regions like the one studied in this report may be a key factor in why the signal recognition particle must be able to bind to a variety of amino acid sequences. In membrane proteins, the first series of hydrophobic residues along the polypeptide chain serves as the signal sequence for insertion of the protein into the membrane of the endoplasmic reticulum. In many polytopic membrane proteins, this sequence is not cleaved and becomes one of the transmembrane sequences in the folded protein. The results described above for the aspartate receptor and experiments with other proteins imply the transmembrane sequences within a multi-spanning protein interact intimately. During evolution, as polytopic membrane protein sequences multiplied and diverged to serve many roles in the cell membranes, the sequences of their transmembrane regions also diverged. In order to maintain close packing among transmembrane sequences, all the transmembrane sequences in a protein would have to evolve together, including the first transmembrane sequence. In order for the signal recognition particle to continue to interact with those proteins in which the first transmembrane sequence is the signal sequence, it had to either maintain (or obtain) the ability to interact with a wide variety of signal sequences.
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Acknowledgments |
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Notes |
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References |
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Received April 1, 1999; revised June 15, 1999; accepted June 25, 1999.