Arginine Substituted for Leucine at Position 195 Produces a Cyclic AMP-independent Form of the Escherichia coli Cyclic AMP Receptor Protein*

Mutant forms (CRP*) of the Escherichia coli CAMP receptor protein (CRP) that activate CRP-dependent promoters in the absence of the normal allosteric effec- tor (CAMP) have been described. the properties of three CRP* mutant proteins. One protein, 220 CRP, has amino acid substitutions at positions 127 and 170 and low CRP* activity in vivo. A second protein, 222 CRP, has the amino acid substitutions present in 220 CRP and a third substitution (arginine for leucine) at position 195. 222 CRP has high CRP* activity in vivo and high apparent affinity for lacP DNA relative to the 220 CRP in vitro. In this report, we evaluate the effect of a single amino acid substitution at position 195 (leucine to arginine) on CRP activity both in vivo and in vitro.

protein, 222 CRP, has the amino acid substitutions present in 220 CRP and a third substitution (arginine for leucine) at position 195. 222 CRP has high CRP* activity in vivo and high apparent affinity for lacP DNA relative to the 220 CRP in vitro. In this report, we evaluate the effect of a single amino acid substitution at position 195 (leucine to arginine) on CRP activity both in vivo and in vitro.
Cells (cyaAcrpAl pJH8crpR195) containing R195 CRP were found to exhibit a CRP* phenotype, expressing a variety of CRP-dependent genes in the absence of added CAMP. R195 CRP exhibited both CRP* activity in vitro and increased apparent affinity for cAMP relative to wildtype CRP. CRP titration experiments performed using an in vitro lac transcription system suggest that the isolated substitution of arginine at position 195 does not confer on CRP the high lacP affinity that distinguishes the 220 and 222 forms of CRP. These findings lead us to the conclusion that the effects of multiple mutations in CRP can be both cumulative and interactive.
The cyclic AMP receptor protein (CRP)' modulates the rate of mRNA synthesis at a specific class (CRP-dependent) of promoter (reviewed in Ref. 1). In wild-type Escherichia coli, the activity of CRP-dependent promoters is controlled by the cellular concentration of CAMP which establishes the concentration of the active CRP.cAMP complex form of CRP (2). When bound to the appropriate site on DNA, CRP-cAMP enhances RNA po1ymerase:promoter recognition. At least three mechanisms exist to increase CRP-dependent promoter activity in cells unable to synthesize cAMP de novo ( i e . cya-).
The first involves mutation of a specific promoter to a CRPindependent class (3); the mutation affects the synthesis of mRNA from only the mutant promoter while all other members of the CRP-dependent promoter class remain dependent upon the CRP SCAMP complex for activity. The second mechanism involves mutation of RNA polymerase such that the enzyme exhibits increased recognition of some CRP-dependent promoters (4)(5)(6). The third mechanism results from mutation of the crp gene where many, if not all, CRP-dependent promoters are relieved of the normal cAMP requirement yet remain dependent upon mutant CRP.
Mutant strains of E. coli containing forms of CRP that function independently of cAMP (CRP*) have been isolated (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). We have recently described the genetic and biochemical characteristics of three forms of CRP* establishing: 1) the sites of mutations in crp; 2) the effects of these mutations on CRP* structure and effector specificity; and 3) the requirements of these forms of CRP to activate wild-type lacP in vitro (19). One such mutant protein, 220 CRP, has amino acid substitutions at positions 127 and 170 (19). A second mutant protein, 222 CRP, has the amino acid substitutions present in 220 CRP and an additional substitution of arginine for leucine at position 195. The results of protease digestion studies indicate that the conformations of these two forms of CRP* are different (19). In addition, 222 CRP activated a greater number of CRP-dependent promoters than did 220 CRP and exhibited increased apparent affinity for lacP DNA in the absence of CAMP. To establish the effect of the substitution at position 195 on the structure and function of CRP, independent of the effects of mutations at positions 127 and 170, we have constructed a mutant crp allele that substitutes arginine for leucine at position 195 and characterized the R195 CRP in vivo and in vitro. This study shows that R195 CRP is itself a CRP* form of CRP. The protein exhibits altered CRP conformation, altered CRP effector specificity and increased affinity for cAMP relative to the wild-type protein. Materials pKL201, pHA7, pJH4, pJH6, and pJH8 were isolated by the procedure of Norgard (25). The final step in purification was buoyant density centrifugation through CsClz gradients for plasmids pKL201 and pHA7 and chromatography on Sepharose 4B following RNase treatment and phenol extraction for plasmids pJH4, pJH6, and pJH8. Restriction enzymes, T4 DNA polymerase, T4 DNA ligase, and the large fragment of DNA polymerase I (Klenow) were obtained from New England Biolabs, International Biotechnologies, and Bethesda Research Laboratories. NACS Prepac columns were supplied by Bethesda Research Laboratories. These materials were used according to the protocols supplied by the manufacturers. RNA polymerase was supplied by New England Biolabs. Placental RNase inhibitor (RNasin) was supplied by Promega Biotec. Nucleoside triphosphates were purchased from Sigma (ATP) and Pharmacia LKB Biotechnology, Inc. (GTP, CTP, and UTP). [c~-~*P]UTP (800 Ci/mmol) was purchased from Du Pont-New England Nuclear. Dideoxynucleotides were purchased from P/L Biochemicals. Synthetic oligonucleotides were provided by Dr. Gerald Zon of the Food and Drug Administration. All other chemicals were reagent grade.

PhogelPhrnid Shuttle System
To facilitate the in vitro mutagenesis of the wild-type crp allele and the subsequent expression of the mutant crp allele, the following phage/plasmid shuttle system was developed Step I-The cohesive ends of a HindIII/EcoR I M13mp18 DNA digest were made blunt with the addition of the appropriate deoxynucleoside triphosphates and the Klenow fragment of DNA polymerase I. This DNA was cloned into the HpaI site of pKC30. Recombinant plasmids containing the polylinker of M13mp18 were identified first by restriction nuclease digestion of plasmid minipreparations (26) and subsequently confirmed by DNA sequence analysis. One such plasmid, pJH4, contains a tandem insert of the polylinker with the orientation: Step 2-The 942-base pair AluI fragment of pHA7 containing the crp structural gene was isolated from an agarose gel, purified over a NACS Prepac column, and cloned into SmaI-linearized pJH4. pJH4crp-1, contains crp in the sense orientation with respect to the PL promoter and lacks the SmaI.KpnI.SacI.EcoRI.SacI.KpnI.SmaI restriction sites present in pJH4.
-crp-. BumHI.Xba1 fragment of pJH4crp-1 was isolated from an agarose gel, purified over a NACS Prepac column, and cloned into the XbaI site of M13K19. Ml3KlScrp-1 served as template DNA in the oligonucleotide-directed mutagenesis of crp as described below.
Step 4-The double stranded replicative forms of M13K19crpR195 and M13KlScrpP195 were digested with XbaI and cloned into the XbaI site of pJH4. The ligation mixture was used to transform (27) strain C600 (AcI'). AMP' cells were isolated, plasmid minipreparations were made, and recombinant pJH4 were identified by sizing on an agarose gel. Two forms of pJH4crpR195 and pJH4crp-Pl95 were isolated; those that contained crp inserts in the "sense" orientation with respect to the transcript originating at PL and those that contained crp in the opposite orientation. Each plasmid was used to transform strain CA8445/pRK248. Cells containing the sense-oriented crpR195 insert were found to ferment lactose in the absence of added cAMP when cultured at 37 "C on lactose MacConkey agar that contained 50 pg/ml ampicillin and 10 pg/ml tetracycline. This phenotype is characteristic of CA8445/pRK248 that contains a crp* allele (19). Cells containing the nonsense-oriented pJH4crpR195 recombinants failed to ferment lactose in the presence or absence of added CAMP. Cells containing the crpP195 insert in either orientation failed to ferment lactose in the presence or absence of CAMP.
Previous studies have shown that the total cellular protein of cells containing pKC30/crp recombinant plasmids was 5-20% CRP by weight when grown for extended periods at 42 'C (19); however, CA8445/pRK248/pJH4crpR195 failed to synthesize appreciable amounts of CRP when grown at this temperature. Similar results were obtained from a clone containing the XbaI crpR195 fragment in pJH6. pJH6 contains a single M13mp18 polylinker cloned into the HpaI site of pKC3O in the orientation: P,. . . . .SphI.PstI.SulI.XbaI.BurnHI.SmaI.KpnI.SacI.
One difference between crp-containing recombinant plasmids derived from pKC30 compared to those derived from pJH4 or pJH6 is the phasing of ribosomes across the vectorlcrp junction that initiate translation at the N gene AUG. To assess the effect of translational phasing on CRP expression from pJHGcrp, XbaI-linearized pJH6 DNA was treated with mung bean nuclease and blunt end-ligated in dilute solution to yield pJH8. pJH8 has a -1 translation phase shift at the modified XbaI restriction site compared to pJHG; this restores the N gene mRNA translation phasing through the crp structural gene in pJH&rp mRNA to that of the original pKC3O crp constructs. The BamHI fragments of pJH4crpR195 or pJH4crp-1 were cloned into BamHI-linearized pJH8 to yield pJH8crpR195 or pJH8crp-1; the ligation mixtures were used to transform strain CA8445/pRK248. The total cellular protein in extracts of CA8445/pRK248/ pJH&rpRl95 grown at 42 "C was approximately 5% CRP by weight.
Mutagenesis of crp Single stranded M13K19crp-1 was isolated (28) and served as template for oligonucleotide-directed mutagenesis as described by Carter et al. (22). Second strand synthesis was primed with both the SEL2 selection primer (22) and with a 25-base oligonucleotide complementary to crp and having 2-base degeneracy at position 13.
DNA Sequencing Mutant M13 were identified by DNA sequence analysis using the dideoxynucleotide chain termination method (30) in reactions con-

TABLE I
Fermentation chracteristics of strain CA8445/pRK248 containing vector, wild-type or crpR195 recombinant p h m i d s Cells were plated on MacConkey (Difco) indicator medium that contained the indicated fermentable carbon source at 1% (w/v) and, where indicated, cAMP at 0.5 mM. The plates were incubated at 37 "C for 24 h. The fermentation response was scored as follows: -, white colonies; +/-, pink colonies; +, pink colonies with red centers; ++, red colonies; GI, growth inhibition. Cells containing the crp-1 allele cloned into pKC30 exhibited growth inhibition on medium containing xylose and cAMP and fermented maltose in the presence of cAMP (19). This was not observed in cells containing crp-1 cloned in pJH8. This difference probably results from lower levels of CRP expression from pJH&rp-1 compared to pKC3Ocrp-1 (see "Discussion") and reflects cellular CRP concentration differences on strain phenotype.   (19) as primer. Both crpR195 and crpP195 allelic forms of crp were identified. The entire crp was sequenced using primers LMB2 (31), crp. 5, and crp.4 (19) and shown to be wild-type except for position 195.
CRP Purification CRP was purified from extracts of strain CA8445/pRK248/ pJH&rpR195 grown first at 30 "C on LB medium that contained 50 pg/ml ampicillin and 10 pg/ml tetracycline. At a culture density of A, = 0.6, the culture incubation temperature was increased to 42 "C and maintained at this temperature for 16 h. The purification protocol which includes chromatography on phosphocellulose, hydroxylapatite, and CM-Sephadex has been described (19). The protein was judged 95% pure by electrophoresis on 12.5% polyacrylamide-sodium dodecyl sulfate gels that had been stained with Coomassie Blue.
CRP and Protein Assay CRP was assayed by the ammonium sulfate precipitation assay of Anderson et al. (32) as modified by Puskas et al. (14). Protein was assayed by the method of Bradford (33).

TABLE I1
@-Galactosidase induction in strain CA8445/pRK248 containing vector, wild-type, or mutant crp alleles Overnight cultures were grown at 30 "C in LB medium that contained ampicillin a t 50 pg/ml and tetracycline at 10 pg/ml. Inocula were diluted into 37 'C medium a t Asw = 0.05, grown to A, = 0.5 and induced for @-galactosidase by dilution into prewarmed medium that was 5 mM in IPTG. Where indicated, cAMP or cGMP were present at 1 mM. After 1 h, samples were diluted 1:2 into ice-cold medium that was 200 pg/ml in chloramphenicol. 8-Galactosidase was measured by the method of Miller (41). The data are presented as a fraction of the activity observed in the pJH&rp-1 + cAMP culture (1282 units). Protease digestion reactions were run as described previously (19) using CRP purified through the hydroxylapatite step.

Transcription Assay
Transcription reactions were run, the RNA was analyzed and the lac and rep RNA species were quantitated, as described previously (19), with the modification that RNasin (0.3-0.4 units) was included in each reaction to inhibit RNA degradation. Control experiments showed that RNasin had no effect on the properties of R195 CRPmediated lac mRNA synthesis (data not shown). RNasin did improve transcript stability and reduce the background radioactivity in the gels thereby improving the overall quality of the autoradiograms.

RESULTS
CRP-dependent Promoter Activation in Vivo-Cells canying chromosomal deletions of the cya and crp genes and transformed with the vector plasmid pJH8 exhibited a CRPphenotype on indicator plates and established the base-line sugar fermentation reaction used to assess crp allele effects on CRP-dependent promoter activity (Table I). Cells containing the wild-type crp recombinant plasmid pJH&rp-1 were found to ferment nine sugars only in the presence of CAMP. Cells that contained the recombinant plasmid pJH&rpR195 exhibited a CRP* phenotype. Fermentation in the absence of cAMP was limited to four of the nine sugars tested. The fermentation of ribose, xylose, sorbitol, mannitol, and maltose in strain CA8445/pRK248 containing the crpR195 recombinant plasmid required the addition of cAMP to the medium, whereas fermentation of arabinose, galactose, gluconate, and lactose did not (Table I). This sugar fermentation pattern is identical to that observed in cells that contained the crp220 recombinant plasmid (19).
Further characterization of the R195 CRP phenotype was conducted in liquid medium. IPTG failed to induce B-galactosidase synthesis in cultures of CA8445/pRK248/pJH8 in the absence or presence of added cAMP (Table 11). In cells that contained the wild-type crp recombinant plasmid pJH&rp-1, IPTG induced 8-galactosidase synthesis only in the presence of CAMP; cGMP did not substitute for CAMP. In cells that contained the crpR195 recombinant plasmid pJH&rpR195, IPTG in the absence of cAMP induced only slightly higher levels of @-galactosidase than cells that contained wild-type CRP; however, the addition of either cAMP or cGMP stimulated P-galactosidase synthesis in cells con- taining the R195 CRP. These results indicate that R195 CRP has very low CRP* activity with respect to lac-P activation in uiuo as well as the altered cyclic nucleotide specificity frequently associated with CRP* forms of CRP (Table 11).
Characterization of the R195 CRP in Vitro-Wild-type CRP is relatively resistant to protease digestion in the absence of cAMP and is rapidly degraded by proteases in the presence of cAMP (Refs. 34 and 35 and Fig. 1). This characteristic has been interpreted as evidence of a CAMP-induced change in CRP conformation and correlates with the CAMP-dependent CRP activity in transcriptional control (19,34,35). Previous studies have demonstrated that CRP* forms of CRP are more seilsitive to proteases in the absence of cAMP than is wildtype CRP (15,19). R195 CRP was degraded, in the absence of CAMP, by subtilisin, trypsin, chymotrypsin, or the Staph-yl~coccus aurew V8 protease, a result consistent with its classification as a CRP* form of CRP (Fig. 1). Control reactions demonstrated the CAMP-dependent protease sensitivity of wild-type CRP (Fig. 1). These data, together with that of  (19). pKL201, the template used in this transcription system, contains a CRP-independent promoter (repP) that initiates the synthesis of a 106/107-base transcript that is resolved in polyacrylamide gels from the 136/137-base transcript originating from lacP (19). rep mRNA levels serve as an internal control by which to measure either wild-type or mutant CRP effects on lacP activity. The results presented in Fig. 2A demonstrate that, unlike wild-type CRP, the R195 CRP activates h P independently of cAMP in vitro in a CRP concentration-dependent manner. These data provide direct evidence for the CAMP-independent function of R195 CRP. The effect of the R195 mutation on the apparent affinity of CRP for cyclic nucleotides has been determined by titrating either cAMP or cGMP in reaction mixtures that contained The substituted amino acid is indicated using the standard one-letter abbreviation. The numbering begins bCRP' is used to indicate an absolute requirement for cAMP CRP* indicates cAMP independence. The e Proteases include subtilisin, trypsin, chymotrypsin, and the S. aureus V8 protease.
with valine as the amino-terminal amino acid.
phenotype indicated was determined on lactose MacConkey indicator medium using strain CA8445/pRK248. Apparent association constants were determined in lac transcription reactions and are assumed to reflect the affinity of CRP for either CAMP, cGMP, or DNA. Reaction conditions were varied to suit the experiment cyclic nucleotide titrations were run under conditions where CRP* activity was minimal (50 nM CRP); CRP titrations were run in the absence of cyclic nucleotide.
'At equilibrium, cAMP and cGMP bind wild-type CRP with similar affinity (42). The CRP.cGMP complex does not, however, activate hcP.  (44). The open circles represent amino acid residues: 61-64, located on @-strand 5; 82-86, located on @strand 7; 120-127, located on a-helix C. Amino acid side chains that interact with cAMP are shown and hydrogen bonds indicated by dotted lines. Note that serine 128 of the adjacent subunit, which also interacts with the N-6 of CAMP, is not shown. 50 nM R195 CRP (Fig. 2B). It was previously shown that transcription reactions containing wild-type CRP require 1 PM cAMP to half-maximally activate lacP (19). The data presented in Fig. 2B demonstrate that R195 CRP has relatively high apparent affinity for CAMP; approximately halfmaximal activation of lacP was observed at a cAMP concentration of 20 nM. In contrast, R195 CRP has lower apparent affinity for cGMP; half-maximal lacP activation required the addition of cGMP to approximately 10 PM final concentration. Table I11 summarizes the properties of CRP* mutants presented in (19) and in this paper. The 91 CRP and R195 CRP show similar properties in uitro with respect to lac transcription in the absence or presence of cyclic nucleotides yet differ in their ability to promote CAMP-independent @galactosidase synthesis in uiuo. Cells containing the R195 CRP have a weak CRP* phenotype (Tables I and 11) while cells containing the 91 CRP exhibit an intermediate CRP* phenotype (19). Semi-quantitative evidence, obtained through measurements of CRP-specific antibody reactivity of crude cell extracts, indicates that the level of R195 CRP in cells containing pJH8crpR195 is lower by a factor of 4-8 than the level of 91 CRP in cells containing pKC30crp91-37 grown under identical conditions (data not shown). This suggests that the strength of the CRP* phenotype is determined, at least in part, by the cellular concentration of CRP*, a conclusion consistent with the observed concentration-dependent activation of lacP by four forms of CRP* in uitro (Ref. 19,Table 111).

DISCUSSION
Detailed structural information is available for the CRP. CAMP complex form of CRP. Similar detail is lacking for the unliganded CRP structure. The results of chemical probes of protein structure and physical measurement of protein radii (see Ref. 1 for a comprehensive listing) clearly show that these forms of CRP differ. Computer analysis of CRP secondary structure based on primary amino acid sequence has led Kypr and Mrazek (38) to conclude that the amino-proximal cyclic nucleotide binding domain of CRP undergoes substantial transition upon binding CAMP. They predict that this transition involves the reorientation of a predominantly a-helix structure to the @-roll structure that characterizes the cyclic nucleotide binding pocket in CRP -CAMP crystals. This structural rearrangement appears to increase the apparent affinity of CRP for CAMP. Observations consistent with this prediction come from two sets of experiments. First, all CRP* forms tested share, in the absence of CAMP, a conformational characteristic of the wild-type CRP -cAMP comp1ex:protease sensitivity. This is not a characteristic of uncomplexed, wild-type CRP. Second, both the 91 and R195 forms of CRP contain amino acid substitutions located outside of the cAMP binding pocket (Fig. 3) and both exhibit high apparent affinity for cAMP (Table 111). This increased apparent affinity is not explained on the basis of direct mutational alteration of protein/cAMP contact sites. Similarly, Aiba et al. (17) have identified and characterized additional CRP* mutants (R148, N141, H053, and C142) that contain amino acid substitutions outside of the cAMP binding pocket. A common feature among these forms of CRP is a decreased cAMP concentration requirement for CRP-mediated lacP activation in uivo relative to that observed for cells that contain wild-type CRP (17). These observations suggest that these mutant forms of CRP have increased affinity for CAMP.
Two forms of CRP* (220 and 222 CRP*) have been described that exhibit affinity for cAMP comparable to that of wild-type CRP yet exhibit protease sensitivity similar to other CRP* forms of CRP and to the wild-type CRPecAMP complex (19). This might argue against a correlation between CRP conformation and affinity for cyclic nucleotide; however, both the 220 and 222 forms of CRP have isoleucine substituted for threonine at position 127. Analysis of the CRP.cAMP crystals has led to the proposal that threonine 127 hydrogen bonds with the N-6 amino group of cAMP (Ref. 39 and Fig. 3). It is likely that the 220 and 222 forms of CRP have lower apparent affinity for cAMP relative to the 91 or R195 forms due to the elimination of this putative protein/effector contact, which we expect would decrease the stability of this interaction. Along these lines, Aiba et al. (17) have described a CRP* form of CRP (F062) that contains phenylalanine substituted for serine at position 62. The apparent affinity for cAMP of this form of CRP* is similar to that of the wildtype protein. CRP serine 62 is in close proximity to the adenine moiety of cAMP in CRP.cAMP complex crystals (Ref. 40 and Fig. 3B). Computer modeling places the aromatic ring of phenylalanine at position 62 inside the cyclic nucleotide binding pocket; this may hinder hydrogen bond formation between threonine 127 and N-6 cAMP and account for the lower affinity of F062 for cAMP relative to other forms of CRP*.* These considerations, along with the observation that no CRP* has been described having relatively high affinity for cGMP, which cannot hydrogen bond with threonine 127 in a manner similar to CAMP, suggest that 1) one consequence of CRP* mutations is rearrangement of the amino-proximal domain of CRP such that the cAMP binding domain approximates that observed in CRP .CAMP crystals, and 2) CRP conformation plays a role in determining the affinity of the protein for cyclic nucleotide. We feel that a proposal describing a specific mechanism by which CRP* mutations mediate these effects is premature in the absence of additional information regarding the structure of unliganded CRP.
Strain 222 was derived from strain 220 by a spontaneous mutation resulting in the substitution of arginine for leucine at position 195. A primary goal of the current study was to arrive at an understanding of the mechanism by which the mutation at position 195 leads to an apparent 10-fold increase in the affinity of 222 CRP for lacP DNA compared to 220 CRP. It was anticipated that the isolated mutation might confer higher affinity for DNA compared to the wild-type protein without affecting the cyclic nucleotide requirement of the protein. Contrary to this prediction, the substitution of arginine for leucine at position 195 in CRP results in a form of CRP having altered conformation, effector specificity, requirements for promoting lacP activity in vitro and increased affinity for cAMP compared to wild-type CRP. An important conclusion to be drawn from this study is that the effects of multiple mutations in CRP can be both cumulative and interactive.
Position 195 is carboxyl-proximal to the DNA sequence recognition helix (F-helix, Ref. 39) of CRP as illustrated in Fig. 3A. R195 CRP contains the most carboxyl-proximal single amino acid substitution identified to date that confers CAMP-independent function on CRP. Another mutant allele, crpP195, substitutes proline for leucine at position 195 (see "Experimental Procedures"). Unlike R195 CRP, P195 CRP does not confer a CRP* phenotype on strain CA84451pRK248. Cells containing P195 CRP failed to ferment any of the sugars listed in Table I1 in the presence or absence of CAMP. We interpret this as indicating that P195 CRP is an inactive form of the protein. Clearly, different amino acid substitutions in CRP in the carboxyl-terminal portion of the protein, specifi-* I. T. Weber, personal communication. cally at position 195, can affect CRP function and response to CAMP; however, not all such substitutions confer a CRP* phenotype.