The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding

The x-ray crystal structure of the cAMP-ligated T127L/ S128A double mutant of cAMP receptor protein (CRP) was determined to a resolution of 2.2 Angstr om Although this structure is close to that of the x-ray crystal structure of cAMP-ligated CRP with one subunit in the open form and one subunit in th e closed form, a bound syn-cAMP is clearly observed in the closed subunit i n a third binding site in the C-terminal domain. In addition, water-mediate d interactions replace the hydrogen bonding interactions between the N-6 of anti-cAMP bound in the N-terminal domains of each subunit and the OH group s of the Thr(127) and Ser(128) residues in the C alpha -helix of wild type CRP, This replacement induces flexibility in the C alpha -helix at Ala(128) , which swings the C-terminal domain of the open subunit more toward the N- terminal domain in the T127L/S128A double mutant of CRP (CRP*) than is obse rved in the open subunit of cAMP-ligated CRP, Isothermal titration calorime try measurements on the binding of cAMP to CRP* show that the binding mecha nism changes from an exothermic independent two-site binding mechanism at p H 7.0 to an endothermic interacting two-site mechanism at pH 5.2, similar t o that observed for CRP at both pH levels. Differential scanning calorimetr y measurements exhibit a broadening of the thermal denaturation transition of CRP*; relative to that of CRP at pH 7.0 but similar to the multipeak tra nsitions observed for cAMP-ligated CRP. These properties and the bound syn- cAMP ligand, which has only been previously observed in the DNA bound x-ray crystal structure of cAMP-ligated CRP by Passner and Steitz (Passner, J. M ., and Steitz, T. A. (1997) Proc. Nafl. Acad. Sci. U.S. A. 94, 2843-2847), imply that the cAMP-ligated CRP* structure is closer to the conformation of the allosterically activated structure than cAMP-ligated CRP. This may be induced by the unique flexibility at Ala128 and/or by the bound syn-cAMP in the hinge region of CRP*.