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

X M Li,J S Krakow

doi:10.1016/s0021-9258(18)47575-8

Abstract

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.

Highlights

The monoclonal antibody 64D1 was found to bility of direct contact between CRP and RNA polymerase inhibit cAMP binding by the cAMP receptor protein when bound to their respective sites on the DNA
The results indicate that thepresence of the lac P' DNA fragment does not stabilize the CAMP-CRP complex
Binding of monoclonal antibody (mAb) 64D1 to its antigenic site in the assembled topographic sites formed by the native folding of large N-terminal CRP domain apparently alters the conforthe protein [31]

Summary

RESULTS

Binding of monoclonal antibodies to complexes containing CRP and 13H]lacP+DNA was carried out in 1.5-ml Eppendorf tubes in a mixture which contained (final volume, 50 pl): 40 mM Tris-HC1 Incubation of the preformed CAMP-CRP-lacP+complex with mAb 64D1 results in a progressive decomposition of the complex so that after 40 min of incubation (Fig. 2, lane three times with wash buffer, 50 pl of a solution containing 10 nM f ) binding of CRP to lac P+has been lost. NM lac P+DNA fragment, and 40 nM RNA polymerase holoenzyme to which was added CRP (where indicated) or the complex formed after incubation (for 10 min a t 37 "C) of CRP and mAb 64D1. 3 nM [3zP]lacP+fragment, 40 nM CRP, and where indicated, 240 nM mAb 64D1, 120 nM RNA polymerase holoenzyme, and the indicated concentration of CAMP.The components added, the order of addition,. - 40 with [3H]N-ethylmaleimide [18].The datapresented in Table I show that immunoprecipitin binds to the immune complex formed between mAb 64D1 or mAb 66C3and [3H]NEM-CRP

Both monoclonal antibodies are of the IgGl class for which

Monoclonal Antibody TIhnahticbAiBtMsinPding by CRP abcdefghijklm

DISCUSSION

MonoclAonnatilbody That Inhibits CAMPBinding by CRP