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J. Biol. Chem., Vol. 276, Issue 14, 11230-11236, April 6, 2001

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The Structure of the T127L/S128A Mutant of cAMP Receptor Protein Facilitates Promoter Site Binding^*

Seung Y. Chu, Maria Tordova, Gary L. Gilliland, Inna Gorshkova, Ying Shi, Shenglun Wang, and Frederick P. Schwarz^§

From the Center for Advanced Research in Biotechnology of the National Institute of Standards and Technology and the University of Maryland Biotechnology Institute, Rockville, Maryland 20850

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 Å. 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 the closed form, a bound syn-cAMP is clearly observed in the closed subunit in a third binding site in the C-terminal domain. In addition, water-mediated interactions replace the hydrogen bonding interactions between the N⁶ of anti-cAMP bound in the N-terminal domains of each subunit and the OH groups of the Thr¹²⁷ and Ser¹²⁸ residues in the C -helix of wild type CRP. This replacement induces flexibility in the C -helix at Ala¹²⁸, 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 observed in the open subunit of cAMP-ligated CRP. Isothermal titration calorimetry measurements on the binding of cAMP to CRP* show that the binding mechanism changes from an exothermic independent two-site binding mechanism at pH 7.0 to an endothermic interacting two-site mechanism at pH 5.2, similar to that observed for CRP at both pH levels. Differential scanning calorimetry 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 transitions 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. Natl. 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*.

The atomic coordinates and structure factors (code 1HW5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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