J. Mol. Biol. (1977) 114, 47-60

Cyclic AMP-mediated Intersubunit Disulfide Crosslinking of the Cyclic AMP Receptor Protein of Escherichia cob ERIC EILEN~

AND JOSEPH S. KRAKOW.$

Department of Biologiud Sciences Hunter College of the City University of New York, 695 Pad Avenue, N. Y. 10021, U.S.A. (Kpceived

25 January

1977)

The protomeric form of the cyclic AMP receptor protein (CRP) of Escherichiu coli is composed of two identical subunits of molecular weight 22,500 and contains two buried and two available cysteine residues. Titration of the two available cysteines with DTNB § eliminates cyclic AMP-dependent DNA binding activity which is regenerated by incubating the modified protein with /?-mercaptoethanol. In the absence of CAMP, the formation of the TNB anion from DTNB and the incorporation of [l”C]TNB into CRP are approximately stoichiometric. In the presence of CAMP, there is an increase in the rate of formation of the TNB anion while the incorporation of [‘%]TNB into CRP is markedly inhibited. These observat,ions are reconciled by the observation that CAMP induces DTNB-mediated disulfide crosslinking of the two available sulfhydryls to produce a species migrat,ing as a 45,000 molecular weight subunit on SDSpolyacrylamide gels. A mechanism is suggested by which an intersubunit, intraprotomer disulfide bond is produced by secondary disulfide interchange after the incorporation of the init,ial TNB group. Based on the observation of CAMPmediated disulfide crosslinking, t’he available cystrines of the DNA binding region are proposed to reside in close proximity as part of an antiparallel F-sheet structure formed by the two carboxyl proximal pnlypeptides when CRP is in the DNA binding conformation.

1. Introduction Positive control of catabolite sensitive operons in Escherichia coli has been shown to be mediated by the binding of the cyclic AMP receptor protein to the promoter in the presence of cAMP§ (d eCrombrugghe et al., 1971; Lee et al., 1974; Majors, 1975). A conformational change in CRP induced by the hinding of CAMP is required for DNA binding by the protein (Krakow & Pastan, 1973 ; Wu & Wu, 1974). When CRP is subjected to proteolysis in the presence of CAMP, a resistant core is produced, designated aCRP, which binds CAMP but does not show CAMP-dependent DNA binding (Krakow & Pastan, 1973). The CRP protomer is composed of two apparently t Present address: Department of Pathology, Nrw York University School of Medicine, New York, N.Y. 10016, $ To whom correspondence should be addressed. § Abbreviations used: CAMP, adenosine 3’,6’-monophosphak; CRP, cyclic AMP receptor tubercidin 3’,5’-monophosphate; protein; cGMP, guanosine 3’,5’-monophosphate; cTuMP, DTP, 4,4’-dithiodiDTNB, 5-6’-dithiobis(2-nitrobenzoic acid) ; TNB, 2.nitro-5.thiobenzoate; pyridine; SDS, sodium dodecyl sulfate; PMSF, phenyl methane sulfonyl fluoride. 47

4x

E. ELLEN

ASI)

J. $. RRAKO\+

identical subunits of 22.500 molecular weight with four cysteine residues per protomer. of which 1.5 to 2 are available to tikation by DTNB and DTP. respectively (Anderson et al., 1971). The aCRP core protomer derived from CRP has been shown to have two subunits of -12,500 molecular weight with t,uo cysteines per prot’omer which react with DTNB only under denaturing conditions (Eilen & Krakow, 1977). The above mentioned studies on the purified CRP define two regions of the protein; one containing the two buried sulfhydryls and CAMP binding domain and the /3 region containing the two available sulfhydryls and the DNA binding domain. We have found that in the reaction of the available SH groups of CRP with the sulfhydryl reagent DTNB (Ellman, 1959), the protein undergoes primary and seondary disulfide interchanges to produce a disulfide bond linking the subunits. This reaction has been characterized in order to study the conformat,ional events in the p region of CRP which is involved in DNA binding.

2. Materials and Methods (a) Cyclic

AMP

receptor protein

The CRP used in these studies was isolated from E. coli diploid in the CRP structural gene (Anderson et al., 1971) by a method t,o be presented elsewhere. (b) Chemicals

CAMP, cGMP, B’AMP, bis(Tris)propane, j%mercaptoethanol, bovine serum albumin, a-chymotrypsinogen, and subtilisin (8.6 U/mg) were purchased from Sigma. cTuMP was a gift from Dr Ira Pastan. DTNB was a product of Pierce. [l%]DTNB (10,580 cts/min per nmol) was obtained from CEA, France. [“H]d(A-T), were prepared with E. coli DNA polymerase (Jovin were products of New England Nuclear.

(1262 cts/min

per nmol) and d(X), and Liquifluor

et al., 1969). L3H]cAMP

(c) Filters Nitrocellulose filters (0.45 pm pore size) were obtained from Matheson-Higgins stored in 20 m&r-TrisHCl (pH 8) containing 50 mM-NaCl. The GF-C glass fiber filters in the [3H]cAMP binding assays were purchased from Whatman. (d) DTNB

and used

titrations

Determinations of protein sulfhydryl groups were carried out by the method of Ellman (1959) using a Beckman Acta III recording spectrophotometer. Titrations with [14C]DTNB were performed by filtration of the incubation mixtures onto nitrocellulose filter discs after the addition of O-75 ml of wash buffer (20 mix-Tris-HCl (pH S), 50 mM-NaCl). The filters were washed with 2 ml of wash buffer, dried, and counted in Liquifluor/toluene in a Beckman LS230 scintillation counter. (e) Binding

Assays for the binding previously

described

of [3H]cAMP

(Krakow

& Pastan,

(f) Sedimentation

assays

and [aH]d(A-T),

velocity

(R)

Protein

by the method as a standard.

out as

centrijugation

Sucrose density-gradient sedimentation velocity carried out at 4°C in a Beckman L3-50 ultracentrifuge

Protein was determined line bovine serum albumin

to CRP were carried

1973).

studies (Martin & Ames, using an SW-50 rotor.

1961) were

determination of Schaffner

& Weissmann

(1973) using crystal-

nAMP-MEDIATED

DISULFIDE

(h) Polyacrylamide

CROSSLINKING

49

gel electrophoreeis

Electrophoresis on SDS-polyacrylemide gels (Laemmli, 1970) was carried out at 5 mA/ cylindrical gel (0.5 cm diam. x 5 cm) or 30 mA/slab gel in apparatus purchased from Hoeffer Inc., San Francisco, Calif., and the Aquabogue Machine Shop, Aquabogue, Long Island, New York, respectively. The stained cylindrical gels were scanned in the Beckman Acta III spectrophotometer. Protein peaks were quantit’ated by weighing the peak areas cut, from the recorder paper.

3. Results The modification of the two available sulfhydryls of CRP in the reaction with DTNB result,s in the loss of CAMP-dependent binding of d(A-T), (Fig. l(a)). Incubation of the TNB-modified CRP with /3-mercaptoethanol restores d(A-T), binding activity. The TNB-modified CRP shows an unimpaired abilit’y to bind CAMP (Fig. l(b)). Since the DNA and CAMP binding domains are structurally differentiated (Krakow & Pastan, 1973; Eilen & Krakow, 1977), modification of the available sulfhydryls in the DNA binding domain would not be expected to affect CAMP binding. The action of CAMP in effecting a conformation necessary for DNA binding and proteolyt#ic cleavage suggested that a similar effect of CAMP on the rate of modification of the available sulfhydryls might be observed. The results presented in Figure 2 show a’n apparent increase in the rate of reaction of CRP with DTNB when t’he reaction is carried out in the presence of 0.1 mM-CAMP. This reaction has a k value of 0.27 min-l in the absence of CAMP and 0.75 in 0.1 m&r-CAMP. The rate of sulfhydryl modification was not affected by the addition of O-I mm-cGMP. In this experiment,. the reaction is followed spectrophotometrically by t’he release of the TNB anion. The reaction of protein sulfhydryls with DTNB is such t’hat for each TNB group released, one TNB is incorporated via a disulfide bond with the protein. To determine whether CAMP also increased the rate of incorporation of TNB into CRP, the reaction was carried out with [14C]DTNB using a nitrocellulose filter binding assay. The results presented in Figure 3 show that the presence of CAMP resulted in an apparent inhibition in both the rate and the final amount of [14C]TNB incorporated. At 40 ~M-[~~C]DTNB. the maximum incorporation of [14C]TNB into CRP represented the reaction of 723;, of the available sulfhydryls while that in the presence of cL4MP was only 15%. The inhibition of TNB incorporation observed is limited to CAMP and cTuMP which are also able to effect DNA binding and proteolyt’ic cleavage (Krakow, 1975). The addition of 5’AMP or cGMP had little affect on [14C]TNB incorporation relative to the control lacking nucleotides (Table 1). The concentration of CAMP or cTuMP required for the half-maximal inhibition of TNB incorporation (Fig. 4) is within the same order of magnitude as that required for DNA binding or proteolytic inactivation of CRP with these nucleotides present (Krakow, 1975). The results suggested that the ability of CAMP and cTuMP to depress TNB incorporation map be related to the conformational change in CRP elicited by these ligands. The anomaly between the apparent increase in the rate of TNB release and the depressed incorporation of TNB into CRP with CAMP present could be resolved if the protein in the presence of CAMP undergoes a secondary disulfide interchange with the formation of protein disulfide bonds : R-S-S-NB Such a reaction

would

resolve

+ RSH

+ R-S-S-R

the discrepancy

+ NBS-.

between

the two assay procedures

50

E. EILEN

AND

J. S. KRAKOW

i 0.2

20

I

I

1

I

I

30

40

50

60

70

Time

IO

80

(mln)

(b)

I

CRP (pg) FIG. 1. (a) Loss of d(A-T), binding by CRP after sulfhydryl modification; regeneration of activity with /3-mercaptoethaqol. Two pa-allel rea-tions were carried out at 24”C, one in a 0.5.ml quartz cuvet,te and the other in a polystyrene tube. The ma&ion mixes contained 0.1 M-sodium phosphate buffer (pH 8), 0.1 M-NaCl, 10m5 M-DTNB, and 100 pg CRP in a total volume of 0.5 ml. The absorbance of the reaction mixture in the cuvette was continuously monitored at 412 nm (-O-O-). Portions (15 ~1) were removed at the times indicated and assayed for CAMPdependent r3H]d(A-T), binding (-e-e-). After 20 min, the mixture in the tube was made 10 mM in fl-mercaptoethanol and the incubation continued at 24°C. (b) Prior to the addition of p-mercaptoethanol, portions of 6 and 10 ~1 were removed from the reaction containing DTNB (0) and from a similar mixture not containing DTNB (0) and assayed for [3H]cAMP binding.

used. There could be two possible products: intersubunit disulfide bonds or disulfide bonds formed between CRP protomers. Following incubation with DTNB in the presence or absence of CAMP, the modified protein was analyzed on SDS-polyacrylamide gels (Fig. 5). CRP incubated with DTNB in the presence of CAMP showed a major protein band with a molecular weight of 45,000 with the remaining 15%

CAMP-MEDIATED

DISULFIDE

CROSSLINKING

FIG. 2. Effect of CAMP on reaction of CRP with DTNB. Spectrophotometric were carried out at 24’C and monitored at 412 nm using reaction mixtures vol. 0.5 ml): 90 rg CRP, 40m~-bis(Tris)propane (pH 8), 10e5 M-DTNB (-•-•--) CAMP (--O-O--) as indicated.

51

titrations of CRP containing (final and 1O-4 X.

FIG. 3. Effect of CAMP on reaction of CRP with [W]DTNB. The reaction mixtures contained (final vol. 0.1 ml): 15 pg CRP, 40 mM-bis(Tris)propane (pH 8), [‘Y!]DTNB in the concentrations shown (-e-e--) and 10e4 M-CAMP as indicated (-O-O-). After a 15min incubation at 24”C, 0.76 ml of wash buffer (20 mm-Tris.HCl (pH S), 60 mM-NaCl) was added and the mixtures filtered onto nitrocellulose membranes. The filters were washed with 2 ml of wash buffer, dried, and counted.

52

X.

EILES

AN11

J.

TABLE

Effect of various

nucleotides

S. KRAKOW I

on the incorporation

of [14C]l’NB

Nucleotide

Moles [Y!]TNB incorp./mole CRP

None CAMP cGMP B’AMP cTuMP

0.55 0.14 0.53 0.54 0.13

into CRP

The reaction mixtures contained (final vol. 0.1 ml): 15 rg CRP, 20 mna-bis(Tris)propane buffer (pH 8), IOm5 M-[~~C]DTNB, and 1 ml of the indicated nucleotide. After 16 min at 24”C, 60-~1 portions were removed for filtration and counting as described in Materials and Methods.

4, 0

’ 10-7

I

I 10-6 cNMP

I

I 10-5

!

I 10-4

(M)

FIG. 4. Effect of CAMP and cTuMP on [l*C]TNB incorporation by CRP. contained (final vol. 0.1 ml): 15 pg CRP, 40 mm-bis(Tris)propane (pH concentrations of CAMP (-O-O-) or cTuMP (-•-a-). After 6 min were made 0.6~ 10e4 M in [14C]DTNB and incubated for an additional counting were carried out as described in the legend to Fig. 3.

The reaction mixtures 8), and the indicated at 24”C, the mixtures 10 min. Filtration and

migrating with a molecular weight of 22,500 corresponding to the CRP polypeptide. The resolution of CRP reacted with DTNB in the absence of CAMP showed that about 75% of the protein migrated as the CRP polypeptide with the remainder corresponding to the 45,000 molecular weight species. Since the incubation of CAMP +DTNB-modified CRP with fl-mercaptoethanol prior to electrophoresis regenerated the CRP polypeptide, it is apparent that the 45,000 molecular weight species corresponds to a disulfide-linked CRP dimer. CRP crosslinked in the presence of CAMP showed an identical sedimentation profile with that of the native, unmodified CRP (Fig. 6). The results are consonant with the formation of a disulfide bond between the CRP subunits within a protomer.

c4MP.MEDIATED

DISULFIDE

CROSSLINKING

DTNB

&---

53

CRP-s A

B

CRP t CAMP -

CRP

t

DTNB -+

CAMP

+ CAMP -+

DTNB

I CRP-s

4 C

CRP

-CRP

J ME

FIG. 6. Effect of CAMP on crosslinking of CRP subunits. Reaction mixtures containing vol. 50 ~1) 6 H CRP and 30mM-bis(Tris) propane (pH 8) were given two 15-min incubations with 1O-3 M-CAMP and 10V6 M-DTNB as follows. Sample A B C

Incubation CAMP DTNB CAMP

I

Incubation DTNB CAMP DTNB

(final at, 30°C

II

Sample C was incubated for an additional 30 min with 40 mM-/%mercaptoethanol. were then made 10% in sucrose and 0.1% in SDS @al vol. 0.1 ml) and incubated followed by electrophoresis on 12.5% polyacrylamide gels. The gels were scanned Materials and Methods. ME, fi-mercaptoethanol.

The samples 10 min at 6O’C as described in

The lack of dependence on CRP concentration for crosslinking was in keeping with this proposal (results not shown). The formation of a &sulfide bond mediated by DTNB requires one TNB-substituted and one free and proximal cysteinyl residue for disulfide interchange. Conditions favoring modification of both available sulfhydryls should lower the extent of crosslinking. The data presented in Figures 7 and 8 show that the response to increased DTNB concentration is an expected increase in the amount of TNB incorporated and a concomitant decrease in the amount of disulfide bond-crosslinked CRP. The effect of CAM? is in enhancing the extent of intersubunit crosslinking at all DTNB concentrations used (Fig. 8) relative to the control lacking CAMP. This suggests that

Fraction

number

FIQ. 6. Sucrose density-gradient centrifugation of unmodified and crosslinked CRP. A sample (final vol. 0.1 ml) containing 100 pg CRP, 40 mM-bis(Tris)propane (pH 8), 10m4 M-CAMP, and 0.2 x 10da M-DTNB was incubated 20 min at 24°C and adjusted to a sucrose concentration of 1 o/0 (-O-O-). The resulting protein was judged to be 75% crosslinked on SDS-polyacrylamide gels. Another sample (final vol. 0.1 ml) containing 100 pg CRP in 40 mm-bis(Tris)propane (pH 8) was treated similarly but without DTNB (-a-a-). Each sample was layered on a 6% to 20% sucrose gradient containing 10 m&r-potassium phosphate (pH 7.5). Centrifugetion was carried out at 45,000 revs/min for 12 h. Fractions (0.26 ml) were collected and protein determinations performed as described in Materials and Methods.

CAMP induces conformational transitions such that the available cysteinyl residues are brought into the close proximity required for secondary disulfide interchange. The formation of disulfide-linked CRP subunits mediated by DTNB in the absence of CAMP is interesting in light of the proposal by Wu & Wu (1974) that CRP exists in an equilibrium between active and inactive conformational states. As shown in Figure 9, optimal disulfide crosslinking of CRP by DTNB occurs when CAMP is present (Fig. 9 (b) and (c)). W e h ave been unable to identify the minor band in Figure 9(b). Crosslinking is also observed when the ternary complex of CRP with CAMP and d(I-C), is titrated with DTNB (Fig. 9(d)). Similar results were obtained with d(A-T), (data not shown). The aCRP core, which is produced by proteolysis of CRP in the presence of CAMP (Fig. 9(f) and (g)) can also be generated from crosslinked CRP under the same conditions (Fig. 9(h)). These results indicate that the buried sulfhydryls present in the LYregion of CRP are not involved in forming the intersubunit crosslink.

4. Discussion A proposed scheme for the primary and seconary disulfide exchange reactions of CRP with DTNB is shown in Figure 10. Only the available SH groups are depicted, one on each subunit. When DTNB titration is carried out in the absence of CAMP, each sulfhydryl is assumed to incorporate TNB at the same rate with the liberation of a total of two moles of TNB anion. In the presence of CAMP, the protein assumes a conformation in which the available cysteines are brought into close proximity.

CAMP-MEDI.4TED

FIG.

7

DISULFIDE

[‘?I

DTNE

CROSSLINKING

55

CM I

IO0 -

-2

20 --

FIG.

8

PC]DTNB

(M I

FIGS. 7 and 8. Dependence of crosslinking and TNB incorporation on DTNB concentration. The reaction mixtures contained (final vol. 0.1 ml): 15 pg CRP, 20 miw-bis(Tris)propane (pH 8), and t,he indicated concentrations of [‘W]DTNB (Fig. 7) and 10-s M-CAMP (Fig. 8). After a 15-min incubation at 24”C, SO-$ portions were removed for filtration on nitrocellulose membranes and counted as described in Materials and Methods (-O-O-). 50-~1 samples were Th e remaining brought to 20% sucrose and 0.1% SDS in a final volume of 0.1 m land incubated at 60°C for 10 min. Portions (60-~1) were removed from this mixture for electrophoresis on cylindrical 10% polyacrylamide gels. The protein bands were quantit,ated as described in Materials and Methods (-0-O-l.

In this form, t(he incorporation of one mole of TNB is followed by a rapid secondary intersubunit disulfide interchange to form a disulfide bond with the liberation of the incorporated TNB group. The stoichiometry for either pathway would be the same with the release of two moles of TNB anion although only one DTNB molecule would be utilized in the CAMP-mediated reaction to produce the crosslinked CRP. The formation of disulfide bonds through disulfide interchange was originally postulated by Fernandez-Diez et al. (1964) and has been found to occur with rabbit muscle

cAMP-MEDIATED

DISULFIDE

Model

for effect

CROSSLINKING

of CAMP on CRP mtersubunit

57

crosslmklnq

S-S-TNE!

SH

S-S-TNB

SH

t CAMP

FIG.

10.

Proposed

pathways

for

the

reaction

of DTNB

with

CRP.

pyruvate kinase (Flashner et al., 1972), rabbit skeletal muscle tropomyosin (Lehrer, 1975), human platelet membrane proteins (Ando & Steiner, 1976), and the brainspecific protein S-100 (Calissano et al., 1976). The spectrophotometric observation of monophasic, apparent first-order kinetics in the reaction of CRP with DTNB must be considered in light of the crosslinking reaction described above. According to the model, the incorporation of the initial TNB group is slow relative to the secondary CAMP-mediated disulfide interchange reaction Since the stoichiometry of TNB- production is ultimately the same whether the end product is (TNB),CRP or cross-linked CRP, the observed rates of TNBrelease can be considered as composites depending on the equilibrium between the CAMP-bound and non-CAMP-bound forms of the protein (Wu & Wu, 1974). Without CAMP, the observed rate approaches that of the titration of the two available cysteinyl residues. The crosslinking reaction is facilitated by the approach of the available SH groups FIG. 9. SDS-polyacrylamide gel electrophoresis containing 20 pg CRP in 20 mM-bis(Tris)propane with 1 mM-CAMP, 0.1 mnr-DTNB, 20-rg d(I-C),, Sample

of crosslinked CRP and derived at pH 8 (final vol. 0.1 ml) were and 1 pg subtilisin a? follows.

Incubation

I

(a)

-

(b) (0)

CAMP

Cd)

CAMP,

d(W),

(f)

CAMP CAMP,

DTNB

(9) (h)

Sample (e) contained a mixture of 10 pg (final vol. 0.1 ml) in 20 mM-bis(Tris)propane containing subtilisin were made 0.1 rnM SDS and 20% sucrose (final vol. 0.1 ml) layered on a 12.5% polyacrylamide slab ials and Methods.

each

Incubation

aCRP. incubated

Samples at 30°C

II

DTNB DTNB DTNB Subtilisin Subtilisin Subtilisin

of bovine serum albumin and a-ohymotrypsinogen (pH 8). Following the incubations, the reactions in PMSF. Portions (50 ~1) were then adjusted to 0.1% and incubated for 10 min at 60°C. Samples (16 ~1) were gel and electrophoresis carried out as described in Mater-

58

E.

EILEN

8X-D

J.

S. KRAKOW

when CAMP is bound to CRP. Studies using the sulfhydryl crosslinking reagents 1-4(N,N’-phenylene) dimaIeimide and I-2(N,iV’-phenylene) dimaleimide have suggested significant flexibility in the two polypeptide chains of the /3 region with a distance of 5 to 12 A separating the availa’ble cysteines (Pampeno & Krakow, unpublished results). The S-S bond length of 188 A suggests that the shift in conformation on CAMP binding by CRP results in at least, a 3 A decrease in the distance separating the available sulfhydryls. It has been shown t’hat (TNB),CRP is unable to bind [3H]d(A-T), and that CAMPdependent d(A-T)n binding activity can be regained by removing the TNB groups (Fig. 1). DTNB must also dissociate CRP from the ternary complex with CAMP and d(I-C), prior to or during the crosslinking reaction (Fig. 9) since neither (TNB),CRP nor disulfide-crosslinked CRP (not shown) undergo CAMP-dependent DNA binding. At the present time, we cannot reconcile t,hese findings with those of Wu et al. (1974) who reported no change in transcript,ion of hgal DNA by E. coli RNA polymerase after covalent linkage of the fluorescent probeN-(acetyl aminoebhyl)-l-napthylamine-5 sulfonate tjo the available cysteinyl residues of CRP. This was interpreted as demonstrating the lack of involvement of the SH groups in promoter binding. However, it has recently been shown by diLauro et al. (1976) that CRP-independent transcription of gal messenger RNA can occur in vitro. While the available cysteinyl groups in the /I region of CRP may not function in promoter-specific binding, their conformational response to CAMP and the loss of CAMP-dependent d(A-T), binding act’ivity with DTNB titration suggest that they are involved in some component of CAMP-dependent DNA binding. The loss of DNA binding as a result of disulfide crosslinking or TNB incorporation could be a consequence of a necessary involvement of the sulfhydryl groups in hydrogen bonding to DNA (Gursky et al., 1976a). The presence of the bulky, negatively charged TNB group in the DNA binding domain could block DNA binding by steric hindrance and/or charge repulsion. The inhibition of DNA binding by TNB modification or crosslinking could result from an inability of the altered /3 region polypeptide to assume a conformation necessary for DNA binding. Recent studies on protein-DNA interactions have provided models for possible binding mechanisms of regulat’ory proteins to the control region of the lac operon. Gabbay et al. (1976 a,b) demonstrated that various oligopeptides assume a singlestranded p-sheet conformation when bound to double-stranded DNA. In the lac repressor, two anti-parallel /l-sheet chains at the N and C termini of each subunit have been proposed t,o contribute t,o operator binding (Chou et al., 1975). Gursky et al. (19763) have introduced a model for this protein in which cohesive N and C termini polypeptide chains would form twisted, anti-parallel @tructures stabilized by hydrogen bonding in the DNA binding conformation. Inducer binding would alter the conformation of this structure destabilizing hydrogen bonds between the repressor backbone and various bases in the operator helix. CRP in the presence of CAMP is digested by trypsin, chymotrypsin, or subtilisin to generate resistant core forms of similar size. The aCRP core binds CAMP but has lost the DNA binding domain (Krakow & Pastan, 1973). It has been shown that substrate polypeptides are bound in a ,&sheet conformation to these proteases (Kraut et al., 1971; Huber et al., 1971). While obviously inferential, this suggests that CRP in the presence of CAMP undergoes a conformational change in which at least part of the DNA binding domain assumes a p-sheet conformation. This p-sheet

CAMP-MEDIATED

DISULE’IDE

C’ROSSLINKING

59

conformation may be involved in DNA binding by CRP as has been proposed for the Zuc repressor (Chou et aZ., 1975). The CRP binding site of the lac promoter has been sequenced and shown to possess a 2-fold axis of symmetry (Dickson et al, 1975). S’mce CRP consists of two identical subunits, the interaction of the polypeptide region comprising the DNA binding domain in an anti-parallel manner would allow for a simple mechanism for recognizing a symmetrical DNA sequence. Thus far, attempts to react CRP or disulfide crosslinked CRP with the lysyl s-amino group crosslinking reagent dimethyl suberimidate have been unsuccessful (Pampeno & Krakow, unpublished results). The lysyl residues do not appear to be close enough for crosslinking, suggesting that the polypeptides comprising the DNA binding domain may be arranged in an anti-parallel fashion. We propose that the approach of the available SH groups of CRP upon CAMP binding is indicative of the formation of an anti-parallel fi-structure in the DNA binding domain. In this conformation, CRP may be readily crosslinked by a disulfide bridge using DTNB or hydrolyzed to form aCRP. In disulfide-crosslinked CRP, the protein could be locked in an incorrect conformation such that the distortion of the /3 region precludes DNA binding. We are grateful to Dr Ira Pastan for supplying the E. coli K12 cells containing episome. We thank Mr Gopalan Nair for his excellent, technical assistance the CRP preparations used in this study. This investigation was supported States Public Health Service Research grant no GM 22619.

the KLF 41 in preparing by United

REFERENCES Anderson, Chem.

W. B., Schneider, A. B., Emmer, M., Perhnan, R. & Pastan, I. (1971). J. Biol. 246, 5929-5937. Ando, Y. & Steiner, M. (1976). Biochim. Biophys. Acta, 419, 51-62. Calissano, P., Mercanti, D. & Levi, A. (1976). Eur. J. Biochem. 71, 45-52. Chou, P. Y., Adler, A. S. & Fasman, G. D. (1975). J. Mol. Biol. 96, 29-45. decrombrugghe, B., Chen, B., Anderson, W., Nissley, P., Gottesman, M. & Pastan, I. (1971). Nature New Biol. 231, 139-142. Dickson, R. C., Abelson, J., Barnes, W. M. & Reznikoff, W. S. (1975). science, 187, 27-35. dilauro, It., Musso, R., Rosenberg, M., decrombrugghe, B., Sklar, J-. & Wrissman, S. (1976). Fed. Proc. Fed. Amer. Xoc. Exp. Biol. 35, 1595. Eilen, E. & Krakow, J. S. (1977). Biochim. Biophys. Acta. In the press. Ellrnan, G. L. (1959). Arch. Biochem. Biophys. 82, 70-77. Fernandez-Diez, M. J., Osuga, D. T. & Feeney, K. (‘. (1964). Arch. Biochem. Biophys. 107,449-458. Flashner, M., Hollenberg, P. F. & Coon, M. J. (1972). J. Biol. Chem. 247, 8114-8121. Gabbay, E. J., Adawadkar, P. D. & Wilson, W. D. (1976~). Biochemistry, 15, 146-151. Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce, S. & Wilson, W. D. (1976b). Biochemistry, 15, 152-157. Gursky, A. V., Tumanyan, V. G., Zasedatelev, A. S., Zhuze, A. L.. Grokhovsky, S. L. & Gottikh, B. P. (1976a). Mol. Biol. Rep&, 2, 413~ 425. Gursky, A. V., Tumanyan, V. G., Zasedatelev, A. S., Zhuze, A. I,.. Crokhovsky, S. I,. & Gottikh, B. P. (19766). Mol. Biol. Repts, 2, 427-434. Huber, R., Kukla, D., Riihlmann, A. & Steigrnann, 1%‘. (1971). Cold Spring Harbor Symp. Quant. Biol. 36, 14P148. Jovin, ,J. M., Englund, P. T. & Bertsch, L. L. (1969). J. Hiol. Chem. 244, 2998-3008. Krakow, ,J. S. (1975). Biochim. Biophys. Actu, 383, 345-350. Krakow, J. S. & Pastan, I. (1973). Proc. Nat. Acad. ki., U.S.A. 70, 2529-2533. Kraut, J., Robertus, J. D., Birktoft, J. J., Alden, R. A., Wilcox, P. E. & Powers, *J. C. (1971). Cold Spring Harbor Symp. Quant. Biol. 36, 117-123. Laemmli, U. K. (1970). Nature (London), 227, 680-685.

E.

60 Lee,

N., Nut.

ElLEiT

ANl)

J.

S. KRAKOW

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