129

Biochem. J. (1992) 285, 129-136 (Printed in Great Britain)

of the epitope/paratope interactions of a monoclonal antibody directed against adenosine 3',5'-monophosphate

Mapping

Norbert NASS,*§ Christiane COLLING,* Matthias CRAMER,t Hans Gottfried GENIESER,$ Elke Elisabeth WINKLER,t Lothar JAENICKE* and Bernd JASTORFFt

BUTT,:

*Institute of Biochemistry, University of Cologne, An der Bottmuhle 2, D-5000, Koln 1, tlnstitute of Genetics, University of Cologne, Ziilpicherstr. 47, D-5000, Koln 1, and tInstitute of Organic Chemistry, Department of Bioorganic Chemistry, University of Bremen, Leobener Str./NW 2, Postfach 330440, D-2000 Bremen 33, Federal Republic of Germany

A series of systematically modified cyclic AMP (cAMP) analogues, including newly synthesized benzimidazole riboturanosyl 3',5'-monophosphates was used to map the essential molecular interactions between cAMP and the monoclonal antibody 4/2C2 (mab 4/2C2) directed against 2'-O-succinoyl cAMP [Colling, Gilles, Nass, Moka & Jaenicke (1988) Second Messengers Phosphoproteins 12, 123-133]. Its paratope binds the purine base in syn conformation by dipole-dipole interactions and hydrophobic forces and/or stacking interactions. The ribose phosphate moiety is recognized by a combination of charge interactions and H-bonds to the exocyclic and the 5'-oxygen atoms and a hydrophobic interaction at the 2'-position. There is no regioselectivity for the exocyclic oxygen atoms. Compared with the known types of binding, mab 4/2C2 thus shows a new combination of molecular interactions which may be the basis of its strikingly specific recognition and binding of the cyclic adenylates. On this account mab 4/2C2 may become an important tool in studies on cAMP metabolism.

INTRODUCTION Cyclic AMP (cAMP) regulates a multitude of cellular events. It is involved in gene control, signal transduction and chemotaxis, and its function as a messenger in many lower and higher organisms is well documented (Schramm & Sellinger, 1984; Gerisch, 1987). Only in angiosperm plants is the role of cyclic nucleotides still questioned (Spiteri et al., 1989), although cAMP and its turnover enzymes, adenylate cyclase, specific phosphodiesterases and binding proteins, have been identified (Amrhein, 1977; Brown et al., 1979, 1981; Francko, 1983). cAMP acts as a signal for different kinds of receptors to activate genes in bacteria, to elicit aggregation in the cellular slime mould Dictyostelium discoideum and to modulate protein activity in higher eukaryotes. Recently we worked out a competitive radioimmunoassay (r.i.a.) based on the highly specific monoclonal antibody (mab) 4/2C2 raised in mouse hybridoma cells against 2'-O-succinoyl cAMP coupled to keyhole-limpet haemocyanin (Colling et al., 1988). In contrast with the well-known immunoassay techniques based on polyclonal antisera (Brooker et al., 1979), mab 4/2C2 has well defined binding properties. This is particularly important when cAMP has to be determined in the presence of other adenylates or cAMP analogues used to mimic cAMP action. In such cases the discriminatory power of available polyclonal sera or the R subunit of protein kinases is not sufficient. We therefore became interested in identifying the unique molecular interactions involved in recognition and binding of cAMP to the paratope that mab 4/2C2 makes so precise. For this purpose we used a test kit of systematically modified cAMP analogues as probes (Jastorff et al., 1981). To date, three types of cAMP binding have been identified by this technique: the paradigmatic catabolic activator protein '(CAP)-type' from Escherichia coli (Scholuibbers et al., 1984), the

'Dictyostelium cell-surface-receptor type' (see van Ments-Cohen & van Haastert, 1989) and the general 'protein kinase type' (De Wit et al., 1984; Yagura & Miller, 1981). Cyclic-nucleotidespecific phosphodiesterases (cPDEs) from mammals, Dictyostelium and yeast exhibited different molecular interactions depending on the type of protein tested (Erneux et al., 1984; Kesbeke et al., 1985; Braumann et al., 1986; van Lookeren Campagne et al., 1990). A cAMP-binding protein of unknown function has been purified from the green alga Volvox carteri and, when scanned by the test-kit approach, shows only slight similarities to the other known binding types (Feldwisch, 1991). We report here on the results of probing the cyclic nucleotide specificity of mab 4/2C2 (Colling et al., 1988). Our results show it to exhibit a novel and unique type of a very selective cAMPbinding site. It permits the determination of this cyclic nucleotide in the presence of several-orders-of-magnitude higher concentrations of cAMP agonists and antagonists commonly used as specific tools in studying cyclic nucleotide metabolism in cell cultures (Katsaros et al., 1987). MATERIALS AND METHODS

Cyclic [5',8-3H]AMP (1.5-2.2 TBq/mmol) was obtained from Amersham Buchler, Braunschweig, Germany. The nucleotides cyclic AMP, cyclic IMP and cyclic GMP (cAMP, cIMP and cGMP), NM-monobutyryl-cAMP (see Table 2 below for nucleotide abbreviations), 8-Br-cAMP, 8-N3-cAMP, 6-Cl-cPuMP, 2'deoxi-cAMP, 2'-O-succinoyl-cAMP were obtained from Sigma Chemie G.m.b.H., Deisenhofen, Germany. 8-Cl-cAMP, cAMPS [(Rp) and (Sp) isomers] were purchased from BIOLOG Life Science Institute, Bremen, Germany. 8-pCPT-cAMP was obtained from Boehringer Mannheim G.m.b.H., Mannheim, Germany, 8-HIP-cAMP was generously given by Dr. D. Shugar

Abbreviations used: mab, monoclonal antibody; CAP, catabolite activator protein; cPDE, cyclic-nucleotide-specific phosphodiesterase; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; CSR, cell-surface receptor; Krw, relative lipophilicity; cAMP, cyclic AMP (other nucleotide abbreviations are defined in Table 2); r.i.a., radioimmunoassay; IC50 is defined in the text. RI-A, -B, protein kinase regulatory subunit binding sites A and B. § To whom correspondence should be addressed. Vol. 285

130

N. Nass and others

CH, N

36

N

N

NO, 35 -N

N.

NH2

l,343

'

3

31

21

12

NO2

18, 19

C3H\ 023

1o \ C-CH2CH2-CO2

C\

20

--O

-

Cl

13

.1

N-CH3 2

17

1

0

Cl

25 26 27

NI, 26

NH2

.

2

NH/ 1 s

0

CH

0

1

2

I ~~~~~~" 1HO 9, 15, 26

10, 14,27,24

C

30

ci, 16

Fig. 1. Chenical structures of cAMP and cGMP analogues Compound numbers shown in bold are defined in Table 2. The difference between axial (SP) and equatorial the lower left part of the Figure.

Table 1. Chromatographic conditions and yields of the preparation of the new cAMP analogues 33-37 and the phosphothioates 26 and 27 The column contained LiChroprep RP18. Abbreviation: TEAF, triethylamine of given molarity titrated with formic acid to pH 6.5.

(Rp) sulphur substitution is shown in

120 100

a9 80 Flash-chromatography

._

conditions

--a60 (0

Analogue

[Acetonitrile]

[TEAF]

no.

(%)

(mM)

Yield (%)

26, 27 33 34 35 36

15 12 25 17 17

100 10 10 10 10

7, 14

sccco 40

5

51 44 10

(Institute of Biophysics, University of Warsaw, Warsaw, Poland). 8-I-cAMP and 3'-NH-cAMP were obtained from M. Morr (Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany), 2'-0-2,4-dinitrophenyl-cGMP from G. Petridis, 2-NH2-cPuMP, 2-CF3-cBIMP from E. Butt, N6-DimethylcAMP from W. Dostmann, 8-pCTP-cGMP from S. Schulz (all of the University of Bremen). N1-O-cAMP, 2'-0-2,4-dinitrophenyl-cAMP, 5'-NH-cAMP, (Sp)- and (R,)-cGMPS, Sp-6-SEtcPNPS, 7-deaza-cAMP, cPuMP and (Rp)- and (S,)-8-Cl-cAMPS were synthesized by a general cyclophosphorylation method starting from the corresponding nucleoside (Genieser et al., 1988, 1989). The other new analogues were prepared as described below. 5-Nitrobenzimidazole and 5-methylbenzimidazole were purchased from Aldrich (Steinheim, Germany), benzimidazole, 5,6-dimethylbenzimidazole, hexamethyldisilazane, trichlorosilane from Sigma (Deisenhofen, FRG), and Triflate and 1-0acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose from Fluka (Buchs, Switzerland).

-8

-7.5 -7 -6.5 -6 -5.5 log {[cAMP or analoguel(M))

-5

-4.5

Fig. 2. Competition of cAMP and five analogues with 15',8-3HIcAMP for the binding site of mab 4/2C2 Bound radioactivity in the absence of cAMP or derivatives (usually 100-150 Bq) is set to 100%. *, cAMP; A, (SD)-cAMPS; *, (Rd)cAMPS; 0, cIMP; A, Ni-dimethyl-cAMP; EO, 2'-deoxy-cAMP.

The benzimidazole nucleobases, when not commercially available, were synthesized according to published procedures (Kazimierczuk & Shugar, 1989). The benzimidazole 1-fl-Dribofuranosides were prepared as described by Vorbruggen et al. (1981) and Kazimierczuk et al. (1981), then phosphorylated and cyclized by a 'one-pot' method (Genieser et al., 1989). The following new nucleotides were prepared (numbers are those used in Fig. 1 and Table 2 below): 5,6-difluorol-f-D-ribofuranosylbenzimidazole 3',5'-monophosphate (5,6cDFBIMP, 33); 5,6-dichloro-1-,8-D-ribofuranosylbenzimidazole

3',5'-monophosphate(5,6-cDCBIMP,34) ;5,6-dinitro- l-fl-D-ribofuranosylbenzimidazole 3',5'-monophosphate (5,6-cDNBIMP,

35); 5,6-dimethyl- I-,8-D-ribofuranosylbenzimidazole 3',5'-monophosphate (5,6-cDMBIMP, 36). 1992

Cyclic adenylate-binding site of a monoclonal antibody

131

Table 2. Half-inhibition values (IC50), relative free binding energies (6AG') and relative lipophilicity (log K',,) of cAMP and cGMP analogues used in the present study The IC50 values were determined as described in the Materials and methods section. The relative binding energy was calculated from the IC50 values measured in the particular experiments. Each value represents the average result from two independent experiments. The relative lipophilicity of an analogue compared with cAMP (log K',) was determined as described by Braumann & Jastorff (1985).

IC50 Name

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Abbreviation

Adenosine 3',5'-monophosphate Adenosine N'-oxide 3',5'-monophosphate 6-Chloropurineriboside 3',5'-monophosphate 7-Deaza-adenosine 3',5'-monophosphate 8-Bromoadenosine 3',5'-monophosphate 2'-Deoxyadenosine 3',5'-monophosphate 3'-Deoxy-3'-aminoadenosine 3',5'-monophosphate 5'-Deoxy-5'-aminoadenosine 3',5'-monophosphate (Rp)-Adenosine 3',5'-monophosphothioate

cAMP

Inosine 3',5'-monophosphate

cIMP 2-NH2-cPuMP

(Sp)-Adenosine 3',5'-monophosphothioate

2-Aminopurineriboside 3',5'-monophosphate Guanosine 3',5'-monophosphate (Sp)-Guanosine 3',5'-monophosphothioate (Rp)-Guanosine 3',5'-monophosphothioate 8-p-Chlorophenylthioadenosine 3',5'-monophosphate 8-p-Chlorophenylthioguanosine 3',5'-monophosphate 2,4-Dinitrophenoxy-2'-adenosine 3',5'-monophosphate 2,4-Dinitrophenoxy-2'-guanosine 3',5'-monophosphate 2'-O-Succinoyladenosine 3',5'-monophosphate Purineriboside 3',5'-monophosphate 6-Dimethylaminoadenosine 3',5'-monophosphate N0I-Butyryladenosine 3',5'-monophosphate (Sp)-6-Thioethylpurineriboside 3',5'-monophosphothioate 8-Chloroadenosine 3',5'-monophosphate (Rp)-8-Chloroadenosine 3',5'-monophosphothioate

(Sp)-8-Chloroadenosine 3',5'-monophosphothioate

8-Iodoadenosine 3',5'-monophosphate 8-Azidoadenosine 3',5'-monophosphate 8-Hydroxyisopropyladenosine 3',5'-monophosphate Benzimidazoleriboside 3',5'-monophosphate 2-Trifluoromethylbenzimidazoleriboside 3',5'-monophosphate 5,6-Difluorobenzimidazoleriboside 3',5'-monophosphate 5,6-Dichlorobenzimidazoleriboside 3',5'-monophosphate 5,6-Dinitrobenzimidazoleriboside 3',5'-monophosphate 5,6-Dimethylbenzimidazoleriboside 3',5'-monophosphate

All products were isolated by reversed-phase flash chromatography (LiChroprep RP18, 25-40 #tm particle size; Merck, Darmstadt, Germany); 8-chloroadenosine 3',5'-monophosphothioate (Rp) and (Sp) diastereomers (26 and 27) were synthesized as isomeric mixtures (Genieser et al., 1988) and purified further by isocratic micropreparative h.p.l.c. or a second reversed-phase flash chromatography. The solvent system varied with the lipophilicity of the compounds and is given in Table 1. All compounds were characterized by fast-atom-bombardment m.s., 'H and 31P n.m.r., and u.v. spectroscopy; compound 33 was, in addition, characterized by 19F n.m.r. Nucleotide bases were analysed by electron-impact m.s., and nucleosides and nucleotides by fast-atom-bombardment m.s. in positive and negative mode. U.v.-visible spectra were recorded in methanol. Concentrations of compounds were determined by their absorption coefficients. The structure of the cyclic phosphate group was proved by site-selective binding to protein kinase types I and II (Genieser et al., 1992) and hydrolysis by cPDE types II and III. The relative lipophilicity compared with cAMP was determined as described by Braumann & Jastorff (1985), dipole moments,

highest-occupied-molecular-orbital (HOMO) and lowest-unVol. 285

N'-O-cAMP 6-Cl-PuMP 7-CH-cAMP 8-Br-cAMP 2'-H-cAMP 3'-NH-cAMP 5'-NH-cAMP

(Rp)-cAMPS (Sp)-cAMPS cGMP

(Sp)-cGMPS (Rp)-cGMPS

8-pCPT-cAMP 8-pCPT-cGMP

cPuMP N6-DM-cAMP

NM-But-cAMP

Sp-6-SEtcPuMPS 8-Cl-cAMP

(Rp)-8-Cl-cAMPS (Sp)-8-Cl-cAMPS 8-I-cAMP

8-azido-cAMP 8-HIP-cAMP cBIMP

2-CF3-cBIMP

5,6-cDFBIMP 5,6-cDCBIMP 5,6-cDNBIMP 5,6-cDMBIMP

6G

(UM)

(kJ/mol)

log K'w

0.1 3.0 0.1 0.2 0.05 0.15 1.5 28 9.5 4.5 12 6.5 100 540 400 0.03 30 0.2 8.2 0.1 0.7 0.3 0.3 3.7 0.2 0.5 1.0 0.08 0.07 0.4 1.5 0.25 0.55 0.1 6.0 0.15

0 5.7 -0.2 0.8 -2.3 1.0 5.7 14.0 10.1 8.3 10.7 10.4 16.0 20.1 20.5 -3.2 13.4 0.5 9.7 -1.2 3.7 1.2 2.4 8.9 1.3 4.1 5.7 -0.6 -0.9 3.1 5.4 2.3 4.2 -1.3 10.1 0.8

1 0.54 1.05 1.08 1.28 0.85 0.9 0.85 1.15 1.23 0.67 1.14 0.62 1.04 0.8 2.5 1.74 2.14

0.92 1.4 1.29 2.27 1.27 1.56 1.91 1.21 1.48 2.59 1.93 2.68 2.0 2.28

occupied-molecular-orbital (LUMO) values of the purine and benzimidazole bases calculated by means of the MOPAC (molecular orbital package) program supplied by J. J. P. Stewart, Department of Chemistry, University of Texas, Austin, TX, U.S.A.) (see Table 3 below). The cyclic nucleotide analogues were checked by reversed-phase h.p.l.c. to be free of cAMP or the corresponding thio-diastereomer less than 0.001 %. Studies on the binding to mab 4/2C2 were performed as described previously (Colling et al., 1988). RESULTS Rationale for the selection of analogues For the mapping of the mab 4/2C2 paratope (Colling et al., 1988), highly specific for intact cAMP compared with cGMP and cAMP metabolites, we assayed a wide range of chemical modifications introduced into the cyclic nucleotide structure. Compounds 2-11, 18, 21 and 31 were selected according to the wellestablished 'test kit' concept (Jastorff et al., 1981). They represent the minimum set of derivatives by which the essential interactions between cAMP and its receptors can be mapped. Compounds

N. Nass and others

132 2

4.0 3.5 3.0

-

-

u2.5 -=

==

1.5 1.0

0.5 0

2

4

6

8 10 12 cAMP (pmol)

14

16

18

20

Fig. 3. Specificity of mab 4/2C2 for cAMP in the presence of 0 (A), 0.1 jaM-

(El), 0.4 /M- (0) (RP)-8-Cl-cAMPS

C. and CO are defined as bound radioactivity with or without added nucleotide, corrected for non-specific binding (no mab 4/2C2 added).

13-19 allow one to probe the discrimination between cAMP and cGMP derivatives. Analogues 18-20 were used to show interactions at the 2'-position. Derivatives 12 and 21-24 assay the involvement ofH-bonding between the 06-NH2 and the antibody. Compounds 5, 25, 28, and 30 are able to delineate the steric situation at position 8 of the nucleotide base and, consequently, the degree to which rotation around the glycosidic bond (syn/anti equilibrium) is restricted. Derivatives 9, 10, 14, 15, 26 and 27 were chosen to investigate interactions with the axial (S0-) or equatorial (Rp-) exocyclic oxygen atoms. In addition, the benzimidazole ribosfuranosyl 3',5'-monophosphates (compounds 31-36) were included to investigate the influence of changes in strength and angle of the dipole moment and the HOMO and LUMO values in homologous derivatives which are not able to form hydrogen bonds. Compounds 5, 9, 10, 16, 23, 25 and 29 are commercially available and are therefore widely used in biochemical and pharmacological studies. The analogues 24, 26 and 27 are cell-membrane-permeable cAMP agonists (Sp) or antagonist (Rp) (Dostmann et al., 1990). Compounds 14 and 15 act as cGMP antagonist or agonist respectively (Butt et al., 1990). It was of interest to know whether their biologically active concentration would interfere with cAMP determination using mab 4/2C2. In order to compare the present results with those obtained with other systems, the relative free binding enthalpies were calculated from the respective IC50 value. IC50 is defined as the concentration of nucleotide which half-inhibits binding of [5',83H]cAMP in the competitive binding assay; hence, it represents the apparent affinity to the mab 4/2C2 paratope of the compound used. The appropriate concentrations are taken from the binding curves (Fig. 2). SAG' is the change in binding energy compared with cAMP calculated as prime from the inhibition constant, Kj(= IC5O(derivative)/IC50(cAMP)) by: 8AG' = RTlnK,

Mapping of the mab 4/2C2 epitope The binding affinities of 36 cyclic nucleotide derivatives, i.e. their IC50 values, are given in Table 2. All compounds studied are able to compete with cAMP for the binding site of mab 4/2C2, but their IC50 values vary within five orders of magnitude. A first class of compounds shows higher affinity than cAMP (5, 16, 20, 28, 29 and 34). The second group, of derivatives, although representing a wide range of modifications, act similarly to cAMP (2-4, 6, 7, 9-12, 18, 19, 21-27 and 30-36). A striking loss

in affinity is found in the third class of analogues, mainly consisting of cGMP (13) and its derivatives (14, 15 and 17). It is particularly interesting that the characteristic functional groups of adenine (06 -NH2 and N1) can be substituted or even removed (3, 21-23 and 31-36) without loss of relative strength of binding to the paratope. The benzimidazole-containing analogues (31-36) have, with the exception of (35), an unexpectedly high affinity to the paratope. This is most pronounced in the rather lipophilic dichloro and dimethyl derivatives (34 and 36). In contrast, the existence of a guanine base diminishes the affinity by a factor of 1000-5000 (compounds 13-15 and 17). Bulky substituents at position 8 (5, 16, 28 and 30) yield similar or even better binding activity. 8-pCPT-cAMP (16) exhibits the highest affinity found in the present study. The corresponding cGMP derivative (17) also binds better than cGMP itself, but cannot overcome the 'negative guanine effect'. The 5'-amino substitution (8) leads to a much stronger reduction in affinity than the corresponding 3'-NH compound (7). Modifications at the 2'-position (6, 18 and 20), even the introduction of the bulky and hydrophobic 2,4-dinitrophenoxy group (18), do not reduce binding activity. The introductions of sulphur in either the equatorial (Rp) or the axial (SP) position of the cyclic phosphate moiety invariably decreases affinity relative to the corresponding oxygen compounds (see 1, 9 and 10 or 25, 26 and 27 in Table 2). The (Rp) derivatives bind slightly better than the corresponding (Sp) diastereomers. Measurement of cAMP in the presence of other nucleotides A protein used for the determination of the cAMP content in cell cultures in the presence of cAMP analogues must have a high selectivity for cAMP. At about equal nucleotide concentrations, the effect of the analogue is negligible if Kj is at least 10. We used the cAMP antagonist Rp-8-Cl-cAMPS (26) with a Kj = 5 at extremely high concentrations to show that the r.i.a. gives a linear and reproducible response even at drug concentrations around the IC50 value (see Fig. 3).

DISCUSSION The essential interactions of cAMP with its various receptor proteins can be estimated by means of a set of cAMP analogues, each one specifically modified to alter the potential to form distinct molecular interactions. By this method a variety of cAMP-binding sites have been characterized. In the present study we show, by the same rationale, that the paratope of the highly cAMP-specific antibody mab 4/2C2 does not correspond to any of the hitherto-established cAMP-binding types. It represents a novel type of molecular recognition of cyclic AMP with the following properties. (1) Binding of the nucleobase The antibody discriminates sharply between cAMP and cGMP (see Table 2). In 7-CH-cAMP (4) and N1-O-cAMP (2), the H-bond acceptors at the nucleobase are substituted without any decrease in affinity. Furthermore, substitutions of 6-NH2 by Cl (3) or an OH function (11) or even removal of the 6-amino group, resulting in cPuMP (21), do not show any significant increases in Kj values. From this we conclude that H-bonding is not involved in the binding of the nucleotide base. However, comparing the calculated dipole moments (Table 3) with the IC50 values (Table 2), there is good agreement between the apparent affinity of the derivative and the size and direction of the dipole moment of the base. With increasing direction of the dipole moment from 42.50 in cAMP to 157.3° in cGMP the affinity decreases. Furthermore, 1992

Cyclic adenylate-binding site of a monoclonal antibody

133

Table 3. Theoretically determined physicochemical properties of the bases of selected nucleotides Size and direction of the dipole-moments relative to the plane of the aromatic ring (origin of the co-ordinate system is C2 of the adenine base), and the HOMO/LUMO values were quantum-mechanically calculated as described in the Materials and methods section. Dipole moment

Compound cAMP (1) cPuMP (21) 2-NH2-cPuMP (12) cIMP (11) cGMP (13) 5,6-cDNBIMP (35) cBIMP (31) 5,6-cDFBIMP (33) 5,6-cDMBIMP (36) 5,6-cDCBIMP (34) 2-CF3-cBIMP (32) 8-Br-cAMP (5) 8-Cl-cAMP (25) 6-Cl-PuMP (3)

Size (Debye)

Direction (0)

HOMO (eV)

LUMO (eV)

2.03 3.10 2.54 4.56 5.73 10.02 3.11 5.33 3.08 4.97 4.00 1.12 0.65 4.34

42.5 92.5 136.0 155.5 157.3 75.0 134.8 93.4 136.5 95.7 260.6 56.4 74.9 105.6

-8.48 -9.85 -8.72 -9.03 -8.56 -10.41 -8.86 -9.20 -8.79 -9.37 -9.39 -9.01 -9.06 -9.79

-0.14 -0.59 -0.20 -0.46 -0.07 -1.66 -0.03 -0.79 -0.06 -0.71 -0.67 -0.40 -0.44 -0.95

a comparison between relative lipophilicity (i.e. log K'; see Table 2) and apparent binding affinity with the disubstituted cBIMP derivatives (33-36) suggests that binding is directly affected by a combination of lipophilicity and dipole moment (see Table 2). The highly lipophilic dichlorobenzimidazole derivative (34) exhibits the highest affinity found within this group, whereas the dinitro derivative (35) has lowest affinity and lipophilicity of the benzimidazole derivatives probed. From this we infer that the paratope possesses a cleft that orientates and binds the base, presumably by dipole-dipole interactions and hydrophobic forces and/or by stacking interactions with the irelectrons.

(2) Determination of the syn/anti conformation Substituents at position C8 of the base restrict the rotation around the glycosidic bond, i.e. bulky substituents stabilize the syn conformation. All C8 derivatives tested (5, 16, 25 and 28-30) show only moderate changes in affinity compared with cAMP (Table 2). Many of them bind even better than does cAMP itself (5, 16, 28 and 29); this can be correlated with increased lipophilicity. Thus the base is obviously bound in syn conformation. (3) Recognition of the ribose-phosphate moiety Comparison of the relative free binding enthalpies (SAG'; Table 2) leads to the following conclusions. The ribose-phosphate moiety seems to be recognized, orientated and bound by a combination of charge-charge interactions and hydrogen bonds to both exocyclic oxygen atoms and to the 5'-oxygen, but not to the 3'-oxygen, because derivative 8 (5'-NH-cAMP), but not 7 (3'NH-cAMP), in which possible H-bond acceptors are modified, and the analogues with sulphur instead of oxygen in the cyclic phosphate structure (9, 10, 14, 15, 24, 26 and 27), all have much reduced affinity. The ionic interaction is not regioselective for either the axial or equatorial oxygen, since both stereoisomers of all the phosphorothioate derivatives exhibit similar affinities (see 9 and 10, 14 and 15, and 26 and 27). Removal of the 2-hydroxy function does not decrease binding strength (2'-H-cAMP, 6), but esterification at this position (20; 2'-O-succinoyl-cAMP) increases affinity. This hydrophobic interaction reflects the immobilization of cAMP during immunization via the 2'-O-succinoyl linker.

Vol. 285

Fig. 4. Critical interactions and hypothetical topography of the binding site of the monoclonal antibody 4/2C2 + / -, Coulomb forces; X))) H, H-bond donor ;-rTr, hydrophobic interactions.

A model for the epitope/paratope interactions is presented in Fig. 4 and is compared with other binding types (Fig. 5) obtained by the same experimental approach, designed to select analogues that are process-specific tools in cell-biological studies. Therefore the knowledge of cyclic-nucleotide-specificity of the cyclic AMP mab 4/2C2 is not only of theoretical interest, but permits the rational estimation of endogenous cAMP in the presence of certain cAMP and cGMP analogues. As shown paradigmatically for the protein kinase A type I antagonist Rp-8-Cl-cAMPS, the r.i.a. gives a linear response for cAMP up to a constant analogue concentration around its IC50. This makes mab 4/2C2 a valuable tool to study the changes in the cAMP content of cell cultures in

134

N. Nass and others

NH /N

NH2

0

~~~~0N N~~~~~~~~~~~~~~~~~S

C)

0~~~~~~~~~~~~~~~~

H

mab 4/2C2

NH2

O N !

H

CAP

H

RI-B \

~

~

I

N

6o \O

A N

NH2

N

NN

0

0

0~~~~ H

Yeast low-Km cPDE

Fig. 5. Models of the essential interactions of cAMP with the active site of four types of cAMP receptor proteins and the yeast low-K. cPDE mab 4/2C2, RI-B (Jastorff et al., 1979; De Wit et al., 1982; Yagura & Miller, 1981) and the yeast low-Km cPDE (van Lookeren Campagne et al., 1990) bind in syn conformation (syn-type receptors). Dictyostelium CSR (van Haastert et al., 1983) and CAP (Scholubbers et al., 1984; Weber & Steitz, 1987) bind in anti conformation (anti-type receptors). Arrows indicate hydrogen-bonding, and /iE indicate ion pairing. Hydrophobic and/or dipole-dipole interactions have been found in all proteins shown here and are not indicated in the Figure.

1992

Cyclic adenylate-binding site of a monoclonal antibody

135

Table 4. Specificity of six cAMP-binding proteins expressed as relative free binding enthalpies (JAG') &AG' values of RI-B and RI-A were taken from De Wit et al. (1984), of CSR in total from van Ments-Cohen & van Haastert (1989), of CAP from Scholibbers (1985), and of cAMP-specific cPDE (yeast) from van Lookeren Campagne et al. (1990). The IAG' values for mab 4/2C2 were determined as described in the Material and methods section.

Analogue

Relative free binding enthalpies

Abbreviation

No.

cAMP

(1)

N'-O-cAMP 6-Cl-cPuMP 7-CH-cAMP 8-Br-cAMP 2'-H-cAMP 3'-NH-cAMP 5'-NH-cAMP

(2) (3) (4)

(5)

(6) (7) (8)

(Sp)-cAMPS

(9) (10)

cIMP cGMP cPuMP cBIMP

(11)

(Rp)-cAMPS

(13) (21) (31)

Mab 4/2C2

0 5.4 -0.2 0.8 -2.3 0.9 5.7 14.0 10.1 8.3 10.7 16.0 4.4 5.4

RI-B

0 6.3 -0.1 4.0 -0.8 20.2 17.1 17.2 15.2 9.2 8.8 12.7 3.7 5.2

RI-A

CSR

CAP

0 5.6 3.6 -0.3 -1.4 20.2 18.1 16.8 15.9 9.4 2.2 11.9 2.0 6.7

0 9.1 14.6 13.3 15.1 5.6 15.2 4.5 10.8 10.8 21.7 22.7 16.0 11.5

0 2.5 >20 -0.2 >20 >20 > 20 >20 5.7 -5.5

0 12.4 22.0 19.4 3.4 5.3 5.3 7.0 18.0

>20

27.0 27.0

cAPDE

5.2

Table 5. Correlation matrix using the R-squared values of the JAG' values of the 'test kit' analogues (see Table 4) tested on five cAMP protein binding Table 5. Coffelation matrix using the R squared values of the otAG' values of the 'test kit' analogues (see Table 4) tested on five cAMP protein binding sites and the yeast low-K, cPDE (cAPDE)

mab 4/2C2 RI-B RI-A CAP CSR

mab 4/2C2

RI-B

RI-A

CAP

CSR

cAPDE

1.00

0.28 1.00

0.18 0.89 1.00

0.05 0.46 0.46 1.00

0.03 0.12 0.20 0.20 1.00

0.12 0.05 0.11 0.39

cAPDE

the presence of high concentrations of various lipophilic agonists and antagonists of cAMP and cGMP.

Comparison of the binding of different cAMP receptors to mab 4/2C2 Comparison of the binding properties of mab 4/2C2 with four established types of cAMP receptor proteins shows it to exhibit a new type of specificity (Tables 4 and 5). (i) The binding of the adenine in syn conformation by strong hydrophobic interactions without H-bonding is similar to protein kinase-binding sites RI-A and RI-B. (ii) The absence of stereoselectivity for the exocyclic oxygen atoms corresponds to the situation found in Dictyostelium CSR (van Ments-Cohen & van Haastert, 1989) and all protein kinase holoenzymes tested to date (Dostmann et al., 1990), but contrasts with the known stereoselectivity found for the cAMP-CAP interactions (axial) (Scholiibbers et al., 1984) for the cAMP-yeast low-Km PDE interactions (equatorial) (van Lookeren Campagne et al., 1990). (iii) The ability to form a hydrophobic interaction at the 2'position and binding of the 5'-oxygen, but not the 3'-oxygen, by H-bonds is exclusive to mab 4/2C2. Conclusion The interactions of cAMP with the binding site of mab 4/2C2 is due to a new combination of molecular interactions that also leads to specific recognition and tight binding of the nucleotide, making it a promising tool in cyclic nucleotide analysis in the Vol. 285

0.44 1.00

presence of large amounts of other (cyclic) nucleotides and nucleoside di- and tri-phosphates. This work was supported partly through Deutsche Forschungsgemeinschaft by SFB 243 and Grants Ja 58/19, Cr 70/2-1, and through Fonds der Biologischen Chemie and Fonds der Chemischen Industrie. We thank Dr. G. Hubner and Dr. H. W. Klein for critical discussion of the manuscript.

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Received 10 September 1991/11 December 1991; accepted 17 December 1991

1992