1HW5

THE CAP/CRP VARIANT T127L/S128A

Summary for 1HW5

• Entry DOI: 10.2210/pdb1hw5/pdb
• Related: 1G6N
• Descriptor: CATABOLITE GENE ACTIVATOR, ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE (3 entities in total)
• Functional Keywords: camp receptor protein, catabolite activator protein (cap) transcription, allostery, camp, cyclic amp mutant, gene regulation
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 48324.61

Authors

Chu, S.Y.,Tordova, M.,Gilliland, G.L.,Gorshkova, I.,Shi, Y. (deposition date: 2001-01-09, release date: 2001-01-17, Last modification date: 2023-08-09)

Primary citation

Chu, S.Y.,Tordova, M.,Gilliland, G.L.,Gorshkova, I.,Shi, Y.,Wang, S.,Schwarz, F.P.
The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding
J.Biol.Chem., 276:11230-11236, 2001

PubMed Abstract: 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 A. 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 N(6) of anti-cAMP bound in the N-terminal domains of each subunit and the OH groups 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 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 Ala(128) and/or by the bound syn-cAMP in the hinge region of CRP*.

PubMed: 11124966
DOI: 10.1074/jbc.M010428200

Experimental method

X-RAY DIFFRACTION (1.82 Å)