©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interactions between the Methylation Sites of the Escherichia coli Aspartate Receptor Mediated by the Methyltransferase (*)

(Received for publication, August 11, 1994; and in revised form, November 4, 1994)

Michael J. Shapiro Demetra Panomitros Daniel E. Koshland Jr. (§)

From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mutations made at and near the methylation sites of the Escherichia coli aspartate receptor were found to affect the methylation rates of the remaining methylation sites. The results supported a model in which the methyltransferase enzyme contacts a residue seven amino acids to the C terminus of a site being methylated. The presence of a negatively charged residue at that position inhibits methylation, whereas a neutral residue has no effect. Methylation sites in the wild type receptor may also influence the methylation of other sites which are 7 residues away through a physical contact with the methyltransferase.


INTRODUCTION

Covalent modification is a common mechanism by which the function of transmembrane receptors is regulated. In many instances, these modifications are responsible for adaptation(1) , in which the response to a constant stimulus is eventually diminished. In Escherichia coli, the chemotactic response to aspartate is mediated by the aspartate receptor(2, 3) , identified as the Tar protein(4) . Methylation of specific glutamate residues in the cytoplasmic region of the aspartate receptor is responsible for adaptation(5, 6) . The steady state level of methylation changes in proportion to the extracellular aspartate concentration, which counteracts the signal generated by aspartate binding and prevents a sustained chemotactic response to a constant stimulus. Methyl groups are added by a cytoplasmic methyltransferase enzyme(7, 8) , and removed by a cytoplasmic methylesterase enzyme(9, 10, 11) .

The methylation sites of the aspartate receptor have been identified as glutamate residues 295, 302, 309, and 491, which are referred to as methylation sites one through four, respectively(12, 13) . Because sites one, two, and three are spaced in seven-amino acid intervals, they were postulated to be on the same face of a single alpha helix(14, 15) . Site three is the most rapidly methylated position, site two is methylated at roughly half the rate of site three, and sites one and four are both methylated at less than a tenth the rate of site three (14) (see Fig. 1). The differences in rate may be related to the differences in the residues surrounding each methylation site. Based on the pattern of residues surrounding the methylation sites of the aspartate receptor and the highly related receptor for serine(16, 17) , it was first proposed that the methyltransferase optimally recognizes sites within the ``consensus sequence'' Glu-Glu*-Xaa-Xaa-Ala-Ser/Thr, in which the starred glutamate is the site of methylation and ``Xaa'' is any residue(14, 15) . The 4 residues composing this consensus sequence should all be on the same helical face. The consensus sequence thus suggests that the methyltransferase contacts residues on the same helical turn as a methylation site and residues on the adjacent turn in the C-terminal direction.


Figure 1: Amino acid sequences surrounding the methylation sites. A stretch of amino acids containing methylation sites one through three and a stretch containing site four are shown. Xxx indicates any amino acid. The boxes encapsulate positions found in seven-amino acid intervals, with down being equivalent to the C-terminal direction. Residue numbers are shown underneath corresponding residues. The encapsulated glutamates are methylation sites, which are designated on the right along with their relative methylation rates. These rates were derived from Table 1and normalized so that the lowest rate equals one.





Analysis of a series of mutant aspartate receptors in which methylation site residues were mutated from glutamates to aspartates (18) suggested that the methyltransferase also contacts a residue seven amino acids to the C-terminal side of a methylation site, extending the recognition sequence to include residues on three helical turns. Given the spacing between methylation sites one, two, and three, it was proposed that site three is contacted by the methyltransferase when site two is methylated and that site two is contacted when site one is methylated. When an aspartate was present 7 residues to the C terminus of a methylation site, no methylation occurred at that position, suggesting that contact with an aspartate causes the methyltransferase to bind in a nonproductive manner. Because a glutamine is found 7 residues to the C terminus of site three and an alanine is found 7 residues to the C terminus of site four (see Fig. 1), contact with these residues, as with glutamate, apparently allows productive binding.

To test the ideas described above, a mutational analysis was extended to include residues outside the set of methylation sites, specifically residues seven amino acids to the C-terminal side of sites three and four. Mutations were also made at methylation sites two and three, which are 7 residues to the C terminus of sites one and two, respectively. The methylation rates of the mutant receptors were then determined. Mutants will be described using the one-letter amino acid code for the residues at methylation sites one through four, respectively. For instance, EEEE represents a receptor in which all four sites are glutamates, and EQEE represents a receptor in which site two has been converted to glutamine. To describe receptors with mutations outside the set of methylation sites, parenthesis will be placed to the C-terminal side of the letter representing the methylation site closest to the mutation. The parentheses will enclose the number of the mutated residue preceded by the one-letter code for the wild type residue and followed by one letter-code for the mutant residue. For example, EEE(Q316N)E represent a receptor in which the glutamine 7 residues from site three (residue 316) has been changed to asparagine.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were purchased from Operon Technologies (Alameda, CA). Reagents for site-directed mutagenesis were from Bio-Rad. Cycle sequencing reagents were from Life Technologies, Inc. Tritiated AdoMet (15 Ci/mmol) was from Amersham Corp. Scint A-XF scintillation fluid was from Packard. V8 protease was from Pierce. Trypsin was from Worthington. Prestained protein molecular weight markers (low range) were from Bio-Rad. A 30-cm Bondclone C(18) reverse-phase column from Phenomenex was utilized. All other materials were reagent grade.

Strains and Plasmids

HCB721, a strain deficient in all chemotaxis genes(19) , was provided by Dr. Howard Berg (Harvard University). All plasmids were constructed by mutagenesis of the pSK2 plasmid as described previously(18) . Codons changed to glutamate, glutamine, aspartate, asparagine, or alanine were specified by GAG, CAG, GAT, AAC, or GCC, respectively.

Methylation Reactions

Cell membranes containing various mutant receptors and the methyltransferase enzyme were prepared and analyzed as described elsewhere(18) . All reactions were performed by mixing equal volumes of a solution containing receptor in 30 mM sodium phosphate, pH 7.0, 1 mM phenylmethylsulfonyl fluoride, and a solution containing tritiated AdoMet (100 µCi/ml) and methyltransferase in 30 mM sodium phosphate, pH 7.0, 1 mM phenymethylsulfonyl fluoride, 2 mM 1,10-phenanthroline, and 5 mM EDTA. Initial rates of methylation (see Table 1) were determined as described elsewhere (18) using 4 µM receptor and a 1 to 200 dilution of methyltransferase extract. Samples intended for proteolysis were allowed to react under these conditions for 2 min. Extensive methylation reactions (see Fig. 2) contained 2 µM receptor and a 1 to 50 dilution of methyltransferase extract and were allowed to proceed for 2 h at 37 °C.



Figure 2: Methylated peptides derived from receptor mutants. Samples of extensively methylated receptors were subject to proteolysis and HPLC. The amount of radioactivity eluting from the column over time was monitored. Numbered peaks represent a methylated peptide containing the indicated methylation site. These assignments were based on retention times as described in the methods section. The peak found at 5 min in each chromatograph was shown to be methanol (13) . The areas of the different peaks are very similar and do not reflect the differing initial rates of methylation of the individual sites (Table 1), suggesting that receptor methylation was nearly complete.



Determination of Methylation Rates at Individual Sites

Samples of receptors methylated under initial rate conditions (see above) were subject to proteolysis followed by HPLC (^1)as described previously(14, 18) . Radioactive peaks were detected using a BetaRam flow-through detector (INUS Systems). The resulting peptide maps were integrated using the Kaleidagraph program (Abelbeck Software) in order to determine the percent of the total methyl groups incorporated at each site. These percentages were then multiplied by the overall initial rates of methylation to determine the initial rates at each site (see Table 1). The average and standard deviation of the total rates were determined from four or more assays and the percent incorporation at each site was determined from the average of at least two assays.

Analysis of Peptide Maps

The chromatographic mobilities of methylation site-containing peptides derived from EEEE were established previously(14, 18) . The mutation in the EEE(Q316N)E receptor does not alter a residue in one of these peptides. The mutations made at sites two and three alter the composition of peptides containing the mutated sites only. The site four-containing peptide is the 20-mer VTQQNASLVQE*SAAAAAALE (E* is site four). The last alanine in this peptide is replaced by glutamine or aspartate in the EEEE(A498Q) or EEEE(A498D) mutant, respectively. In the EEEE(A498E) mutant, a new V8 cleavage site is introduced which alters the site four-containing peptide to an 18 mer ending in E*SAAAAAE. Each of these changes would be expected to make the site four-containing peptide more polar and decrease its retention time on the column, which is apparent for the EEEE(A498Q) (Fig. 2B) and EEEE(A498E) mutants (Fig. 2D). The apparent absence of a site four-containing peptide in the EEEE(A498D) mutant is unlikely to be caused by that peptide having the same chromatographic mobility as one of the other methylation site-containing peptides, each of which is only a 7 mer.


RESULTS

To determine which residues in the aspartate receptor contribute to the recognition of sites by the methyltransferase, mutations were made at methylation sites two and three, which are 7 residues from sites one and two, respectively, and also at Gln and Ala, which are 7 residues to the C terminus of sites three and four, respectively. The initial rates of methylation at individual sites in the various mutant receptors are listed in Table 1. Methylation rates of receptors with aspartate substitutions at sites one and two, as determined previously(18) , are also listed for comparison.

When Ala was mutated to a glutamine, producing the EEEE(A498Q) receptor, only minor changes in the various methylation rates were apparent (Table 1). When Ala was changed to either an aspartate or a glutamate, no methylation at site four was detectable under initial rate conditions, while changes of less than 3-fold occurred at sites one, two, and three (Table 1). These results are consistent with previous findings (18) that the presence of an aspartate 7 residues to the C terminus of a methylation site drastically reduces its methylation rate and suggests that a glutamate may have a similar effect.

To determine if site four in the EEEE(A498E) and EEEE(A498D) receptors could be methylated at all, the methylation patterns of receptors subject to extensive methylation reactions were analyzed (Fig. 2). The reaction conditions were designed to maximize the extent of methylation. No site four methylation was detectable in the EEEE(A498D) receptor, while site four in the EEEE(A489E) receptor was methylated to a similar extent as in the EEEE and EEEE(A498Q) receptors. This suggests that the aspartate substitution at Ala abolished methylation at site four, while the glutamate substitution reduced the initial rate of site four methylation to an undetectable level.

When the glutamine 7 residues to the C terminus of site three (Gln) was mutated to an asparagine, the methylation rate at all four sites decreased (Table 1). Site three did not seem to be affected to a greater extent than the others. Unfortunately, we were not able to evaluate the effect of changing Gln to either glutamate or aspartate because receptors containing these substitutions were not stably expressed, probably due to proteolytic degradation.

Mutation of site three to glutamine in the EEQE receptor caused an increase in the methylation rates of all other positions (Table 1). The mutation of site three to aspartate was previously found to enhance site one and four methylation while nearly abolishing site two methylation(18) . Thus, the two types of substitutions differ primarily in their effects on site two.

Mutation of site two to glutamine in the EQEE receptor caused only minor changes in the methylation rates of the remaining sites (Table 1). Mutation of site two to either asparagine or alanine caused a roughly 4-fold increase in the methylation rate of site one, while causing less than 2-fold changes at the other sites of methylation. It is informative to compare these results to those of substituting an aspartate at site two(18) . In every cases, changes of no more than 2-fold were observed at sites three and four. However, while the aspartate substitution at site two abolished site one methylation, each of the neutral amino acid substitutions had a positive effect on the rate of site one methylation.

Two triple mutants were constructed in which only site one remained a glutamate. When sites two, three, and four were all aspartates, no site one methylation was detectable (Table 1). When site two was instead a glutamine and sites three and four were aspartates, site one was methylated at a rate similar to that of site one in the EEEE receptor. These results are consistent those obtained from analysis of site one methylation in the EDEE (18) and EQEE receptors (Table 1).


DISCUSSION

In essentially all the mutants analyzed here (Table 1), changes occur in the methylation rates of every site. This effect of methylation site substitutions was also observed in a study of the related receptor for ribose and galactose in which alanine substitutions were made(20) . It is likely that these changes in methylation rate occur through several mechanisms, involving alterations in both the structure of the receptor and in the recognition of individual methylation sites by the methyltransferase. The ability of alterations at and near the methylation sites to influence other methylation sites appears to be a general property of the receptor.

In a previous analysis of aspartate substitutions at the methylation sites of the aspartate receptor(18) , it was found that the effects the mutations had on the methylation of remaining sites could be divided into two categories, referred to as ``type A'' and ``type B.'' Both types of effects are also apparent in the results described here (Table 1). Type B effects are defined as an alteration in methylation rate that occurs at all methylation sites. These effects, which are generally changes of less than 3-fold, were postulated to be the result of perturbations in receptor structure. Type A effects are defined as changes in methylation rate occurring at a single methylation site that are of greater magnitude or of opposite sign than effects occurring elsewhere. Type A effects were postulated to result from a perturbation in the recognition of an individual methylation site by the methyltransferase.

An analysis of the results presented here (Table 1) provides evidence that the methylation rate of a site is influenced by the residue seven amino acids away from that site in the C-terminal direction. For example, in the EEEE(A498D) receptor, there is a complete lack of methylation at site four, which we classify as a type A effect, accompanied by changes of roughly 2-fold in the methylation rates of the other positions, which we classify as type B effects. The results obtained with the EEEE(A498E) receptor show that the introduction of a glutamate can also cause both type A and type B effects.

The residue seven amino acids away from a methylation site can exert a positive as well as a negative influence on the methylation of that site. This is best exemplified by the ENEE receptor, in which the substitution of asparagine at site two caused a 4-fold increase in the methylation rate at site one and slight decreases in the methylation rates of sites three and four. Similar results were also seen in the EAEE and EQEE mutants, indicating that the site two substitutions enhance methylation at site one, positioned seven amino acids to the N terminus of the mutations, through a mechanism distinct from the one affecting the other positions.

The appearance of type A effects in many of the mutants described here, and only at sites seven amino acids to the N terminus of a mutation, provides evidence that a residue at this position can influence the binding of the methyltransferase to a particular site. A model for the interaction between the methyltransferase and the aspartate receptor (Fig. 3) can be derived from results presented here (Table 1) and previously(15, 18) . As shown in Fig. 3, when a glutamate or aspartate is present seven amino acids to the C terminus of a methylation site, methylation is inhibited because the geometry of the methyltransferase-receptor complex is constrained. Perhaps this is due to an ionic interaction between a positively charged residue on the methyltransferase and the negative charge on the receptor. Contact with a glutamate inhibits but does not completely prevent the methyltransferase from binding in a productive manner, while contact with an aspartate prevents methylation entirely. The negative influence of aspartate is demonstrated by the lack of methylation at site one or two in the presence of an aspartate substitution at site two or three, respectively(18) , and by the lack of site four methylation in the EEEE(A498D) mutant (Fig. 2). The negative influence of glutamate is revealed by the increase in site one methylation in the EQEE, ENEE, and EAEE mutants relative to site one in EEEE (Table 1), and by the nearly undetectable methylation of site four in the EEEE(A498E) mutant (Fig. 2). Additionally, the high rate of methylation at site three in EEEE relative to sites one and two may be explained by the presence of a glutamine rather than a glutamate 7 residues to its C terminus (Fig. 1).


Figure 3: The interaction between the methyltransferase and the aspartate receptor. The cartoon shows a proposed interface between a portion of the methyltransferase and a portion of the aspartate receptor. The effect of the residue at site two on methylation of site one is shown, but this is meant to illustrate in general how a residue can influence a methylation site positioned seven amino acids away in the N-terminal direction. The methyltransferase contacts the receptor in three places; the active site, indicated by the arrow, is positioned above a site to be methylated; a second binding pocket contacts the rectangle between the methylation sites, which represents the residues found one helical turn from each site; a positive charge in a third binding pocket contacts a residue two helical turns, or seven amino acids, from the site being methylated. When a glutamate is found at site two (A), the methyltransferase is attracted to it, but can still methylate site one. When an aspartate is found at site two (B), the methyltransferase binds in a distorted manner, preventing site one methylation. When an alanine is found at site two (C), the methyltransferase can bind in a manner allowing optimal methylation of site one. The type of binding shown in C would also occur if site two was a glutamine, an asparagine, or possibly a methylglutamate.



According to our model (Fig. 3), when a glutamine, asparagine, or alanine is present seven amino acids away from a methylation site, the methyltransferase may bind in a productive manner. Differences in the effects of these 3 neutral residues are not obvious, but there is some indication that the rate of methylation is higher when an asparagine or alanine, rather that a glutamine, is present. For instance, substitution of a glutamine for the alanine normally found 7 residues to the C terminus of site four caused a decrease in site four methylation through a type A effect (Table 1). Further, site one is methylated at a greater rate in the EAEE and ENEE receptors than in the EQEE receptor. Perhaps glutamine, which has a size similar to glutamate, has some interaction with the methyltransferase, constraining it slightly, whereas the smaller alanine and asparagine residues do not make contacts.

An alanine is present 7 residues to C-terminal side of site four in the wild type receptor, which would be expected to allow an optimal methylation rate (Fig. 1). Despite this, site four is methylated at a relatively low rate, which is probably due to the deviation of the surrounding residues from the consensus recognition sequence for methylation(14, 15) . The near absence of methylation at site four in the EEEE(A498E) mutant may be explained as a combination of the negative influence of the substituted glutamate and of the other residues surrounding site four.

According to the model presented in Fig. 3, the residue seven amino acids from a methylation site lowers the rate of methylation at that position if it is negatively charged, and has no influence on methylation if it is neutral. Thus, the glutamate residues at methylation sites two and three in the wild type receptor may have a negative influence on the methylation rates of sites two and one, respectively. An intriguing possibility is that the conversion of a methylation site to a neutral methylglutamate by the methyltransferase may mimic the effect of mutating a site to a neutral residue. Consequently, methylation of site two or three would be expected to enhance the rate of methylation at site one or two, respectively. Further, given that the results presented here and elsewhere(18, 20) indicate that virtually any substitution at a methylation site influences the methylation rates of the other sites, it seems very likely that the methylation of one site would influence the methylation of the others through one or more mechanisms.

Our model has some interesting implications about the role of receptor methylation in chemotactic behavior. As a bacterium swims up a gradient of aspartate, the number of unmethylated sites on the aspartate receptor decrease as the concentration of aspartate increases(21) . The proportion of receptors with sites two and three available for methylation has been found to decrease especially rapidly(14) . If methylation at site two or three enhances the rate of methylation of site one or two, respectively, it would help the overall rate of receptor methylation to remain nearly constant despite a diminished number of unmethylated sites. This in turn would help the adaptation rate to remain constant across an aspartate gradient. Interestingly, it was found that bacteria exposed to saturating concentrations of maltose, which signals through the aspartate receptor, adapted to aspartate with kinetics similar to those observed when maltose was absent(22) . The steady state level of receptor methylation at the time of aspartate addition was higher when maltose was added first, suggesting that the rate of adaptation is not appreciably influenced by the absolute level of receptor methylation.

Our conclusions, combined with the recognition sequence described previously(14, 15) , suggests that the methyltransferase normally contacts at least three turns of helix and 6 residues in binding to the receptor. A recognition sequence of this length allows simultaneous recognition of two methylation sites. Substrate recognition by casein kinase II and glycogen synthase kinase-3 provide interesting parallels (23, 24) . These enzymes phosphorylate serines and threonines found in close proximity to negatively charged groups. In some cases, phosphorylation at one site introduces the negative charge necessary for phosphorylation of another. Thus, two phosphorylation sites may be simultaneously contacted by the kinase, and the modification state of one site influences the rate of modification at the other.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK09765 and by a National Science Foundation predoctoral fellowship (to M. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, University of California, 229 Stanley Hall(3206), Berkeley, CA 94720-3206. Tel.: 510-642-0416; Fax: 510-643-6386.

(^1)
The abbreviation used is: HPLC, high pressure liquid chromatography.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.