PDBsum entry 1hw5

Gene regulation

PDB id

1hw5

Contents

			Protein chains

					208 a.a. *

			Ligands

					CMP ×3

			Waters ×216

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The structure of the t127l/s128a mutant of camp receptor protein facilitates promoter site binding.

Authors

S.Y.Chu, M.Tordova, G.L.Gilliland, I.Gorshkova, Y.Shi, S.Wang, F.P.Schwarz.

Ref.

J Biol Chem, 2001, 276, 11230-11236. [DOI no: 10.1074/jbc.M010428200]

PubMed id

11124966

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*.



	Figure 1. Fig. 1. Overall schematic representation of the CRP* molecule where green is the subunit in the closed and yellow is the subunit in the open conformation. A and B are orthogonal views of each other. The cAMP ligands are shown as ball-and-stick models. N and C denote the termini, and h indicates the hinge region.		Figure 3. Fig. 3. Coordination of anti-cAMP by CRP and CRP. The yellow trace is the 3GAP structure (11), and the blue trace is the CRP structure. The Ala^128 residue responsible for the different orientation of the open subunit of the CRP* structure is indicated by the sphere in the C -helix. The syn-cAMP observed in only the CRP* structure is shown at the top of the structures.

Secondary reference #1

Title

The structure of a cap-Dna complex having two camp molecules bound to each monomer.

Authors

J.M.Passner, T.A.Steitz.

Ref.

Proc Natl Acad Sci U S A, 1997, 94, 2843-2847. [DOI no: 10.1073/pnas.94.7.2843]

PubMed id

9096308

Full text

Abstract



	Figure 1. Fig. 1. A MOLSCRIPT (4) ribbon drawing of the CAP dimer bound to DNA and the two cAMP molecules (magenta) per monomer, one labeled^ SYN and the other, ANTI. In one monomer, the larger N-terminal domain is yellow, and the smaller C-terminal domain is blue, while^ the DNA half-site bound to it is light gray. The other subunit is green and the DNA bound to it is dark gray. The syn-cAMP lies on the helix-turn-helix and close to the DNA and a loop from the^ N-terminal domain. The DNA sequence of the half-site is 5 -ATGTCACATTAATTGCGTTGCGC-3 .		Figure 2. Fig. 2. Stereo view of the 2F[o]-F[c] "omit" electron density map at 2.2 Å resolution from which the syn-cAMP (ball-and-stick) was first identified. The density, contoured at 1.2 , was computed using experimentally determined phases from 25 to 8 Å and phases from 8 to 2.2 Å derived from the protein and DNA coordinates alone^ before any coordinates of the syn-cAMP were included in the phase^ and amplitude calculation.