PDBsum entry 1hw5

Gene regulation

PDB id: 1hw5

Contents

Protein chains

208 a.a. *

Ligands

CMP ×3

Waters ×216

* Residue conservation analysis

PDB id:

1hw5

Name:

Gene regulation

Title:

The cap/crp variant t127l/s128a

Structure:

Catabolite gene activator. Chain: a, b. Synonym: camp receptor protein. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: crp/cap. Expressed in: escherichia coli k12. Expression_system_taxid: 83333.

Biol. unit:

Dimer (from PQS)

Resolution:

1.82Å R-factor: 0.228 R-free: 0.300

Authors:

S.Y.Chu,M.Tordova,G.L.Gilliland,I.Gorshkova,Y.Shi

Key ref:

S.Y.Chu et al. (2001). The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding. J Biol Chem, 276, 11230-11236. PubMed id: 11124966 DOI: 10.1074/jbc.M010428200

Date:

09-Jan-01 Release date: 17-Jan-01

Protein chains

P0ACJ8  (CRP_ECOLI) -  DNA-binding transcriptional dual regulator CRP from Escherichia coli (strain K12)

Seq: 210 a.a.

Struc: 208 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1074/jbc.M010428200

J Biol Chem 276:11230-11236 (2001)

PubMed id: 11124966

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

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

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*.

Selected figure(s)

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2001, 276, 11230-11236) copyright 2001.

Figures were selected by an automated process.