Abstract
Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.
Highlights
The binding of 3Ј,5Ј cyclic adenosine monophosphate to cAMP receptor protein (CRP)1 activates the transcription of over 20 genes that code for the catabolite enzymes involved in carbohydrate metabolism in Escherichia coli
This is substantiated by NMR measurements on fluorinated derivatives of CRP, which imply that the conformational change involves the hinge region [1], and proton NMR measurements, which show that CRP in solution tightens up and becomes more rigid upon binding of cAMP [2]
The scattering curve for CRP in the presence of cAMP deviates significantly as shown in Fig. 2 from that of the unligated CRP above Q ϭ 0.15 ÅϪ1. This deviation results from a conformational change in CRP upon ligation with cAMP and is in contrast to the coincidence of the desmeared Small angle neutron scattering (SANS) data from solutions of CRP* in the absence and presence of 17.1 mM cAMP shown in Fig. 3. (Aggregation at this cAMP concentration was not observed for the CRP* solutions.) The nearly coincidental curves in Fig. 3 show that CRP*, contrary to CRP, undergoes very little conformational change in the presence of cAMP, as implied by the in vivo activation of transcription by CRP* in the absence of cAMP
Summary
Vol 273, No 32, Issue of August 7, pp. 20001–20006, 1998 Printed in U.S.A. Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements*. The implication is that the asymmetry of the CRP dimer in the crystal lattice is due to crystal packing forces and that the structural change responsible for the activation of transcription is a change from the open to the closed form in solution This is substantiated by NMR measurements on fluorinated derivatives of CRP, which imply that the conformational change involves the hinge region [1], and proton NMR measurements, which show that CRP in solution tightens up and becomes more rigid upon binding of cAMP [2]. The SANS data can be compared with scattered neutron intensity curves generated from the energy-minimized crystallographic x-ray structure of the protein to determine in more detail the conformation of the protein in solution In this investigation, SANS measurements were performed on solutions of CRP in the absence and presence of cAMP and compared with model scattered intensity curves calculated from the energy-minimized x-ray structures of CRP dimer in exclusively the closed and in exclusively the open forms. SANS measurements were performed on solutions of CRP* in the absence and presence of cAMP
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