Cooperative non-specific DNA binding of the N-terminal core of the cyclic AMP receptor protein of Escherichia coli and its modulation by cyclic AMP

The non-specific DNA binding of CRP and its N-terminal core, alpha CRP, to a 298 base pair DNA fragment, in the presence and absence of cAMP, has been studied using the nitrocellulose filter binding technique and analysed quantitatively using the theory of Clore et al. [J. Mol. Biol. (1982) 155, 447-466]. It is shown that both CRP and alpha CRP bind cooperatively to DNA. At an ionic strength of 100 mM and pH 7.5, the intrinsic equilibrium association constant for the binding of alpha CRP to DNA is approximately 10-times smaller than that for CRP, but the cooperativity parameter is approximately 17-times larger for alpha CRP than CRP. cAMP exerts its effect solely on the intrinsic equilibrium constant and does not alter the cooperativity. In the case of alpha CRP, cAMP reduces the intrinsic equilibrium association constant by a factor of 3, in contrast to the case of CRP where cAMP increases it by a factor of 3. The possible location of the DNA binding site present in the N-terminal core of CRP is discussed in the light of crystallographic data on the cAMP . CRP complex [McKay et al. (1982) J. Biol. Chem. 257, 9518-9524].     

Specific binding of cyclic-AMP receptor protein to DNA. Effect of the sequence and of the introduction of a nick in the binding site.

The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal. These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP. The results are in agreement with the recently published crystal structure of the CRP complexed with DNA [Schutz, S.C., Shields, G.C. and Steitz, T.A., Science 253, 1001-1007 (1991)] showing that the 90 degrees bending of the DNA in the complex results from two kinks.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Characteristics of the binding of human C-reactive protein (CRP) to laminin

Human CRP binds to the basement membrane protein laminin in vitro in a Ca2+-dependent manner via the phosphorylcholine (PC) binding site of C-reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti-idiotypic mAbs directed to the TEPC-15 PC-binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP-laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.     

Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis

C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.     

The binding of the cyclic AMP receptor protein to synthetic DNA sites containing permutations in the consensus sequence TGTGA.

The binding of the cyclic AMP receptor protein (CRP) to symmetrical synthetic DNA-binding sites was investigated with a gel-retardation assay. A set of ten different sequences was employed, comprising all base permutations at positions 2, 4, and 5 of the consensus sequence 5'(TGTGA)3'. We show that: (i) CRP has a higher affinity for the completely symmetrical site than towards the lac wild-type site; (ii) base substitutions at position 2 lead to either a complete loss of specific CRP binding (G----C), a reduction in specific CRP binding (G----A) or only marginal effects on specific CRP binding (G----T); (iii) changes at position 4 abolish (G----C; G----A) or reduce (G----T) specific CRP binding; and (iv) base permutations at position 5 reduce specific CRP binding, but never completely abolish it. Thus position 4, and to a lesser extent position 2, in the DNA consensus sequence are the most crucial ones for specific binding by CRP.     

Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications

C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+ -bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca2+ -binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+ -bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+ -bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+ -bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.     

Regulation of complement activation by C-reactive protein: targeting of the inhibitory activity of C4b-binding protein

C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking beta-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the alpha-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.     

C-reactive protein binds to phosphorylated carbohydrates

C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo-hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.     

Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters

Polyacrylamide gel electrophoresis has been used to visualise and quantitate complexes between the Escherichia coli cyclic AMP receptor protein (CRP) and DNA fragments containing the promoter region of either the E. coli galactose or lactose operons. We show that, although CRP binding to the gal fragment is weaker than binding to the lac fragment, in each case, stable complexes are formed between one dimer of CRP and one molecule of DNA. We have examined the effects of a series of deletions and point mutations in the gal promoter region on CRP binding. From the position of deletions and mutations which prevent the formation of stable complexes, we deduce the location and extent of the sequence at the CRP binding site. We show that it covers approximately the same length of sequence as the binding site at the lac promoter. Unlike the lac site, the gal site contains no palindromic sequence. We discuss the importance of symmetry in the sequence at CRP binding sites and the validity of CRP binding consensus sequences which have been proposed.