Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.     

Roles of hinge region, loops 3 and 4 in the activation of Escherichia coli cyclic AMP receptor protein

The cAMP receptor protein (CRP) requires cAMP for an allosteric change and regulates more than 150 genes in Escherichia coli. In this study, the modular half of cAMP receptor protein was used to investigate the allosteric signal transmission pathway induced by cAMP binding. The activation of CRP upon cAMP binding is indicated to be realignment of the two subunits within the CRP dimer. The interaction of loop 3 and Phe136 do not involve in signal transmission.     

Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain.

Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.     

Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli

Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication.     

Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation.

The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP. We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123. The mutations were made in wild-type crp as well as a cAMP-independent crp, crp*. The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon. These results provide genetic evidence to support that E72 and R82 are essential and S83 and R123 are important in the activation of CRP by cAMP.