Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation

Maria A Schumacher,Gerald Seidel,Wolfgang Hillen,Richard G Brennan

doi:10.1074/jbc.m509977200

Abstract

In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser46-P or Crh-Ser46-P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser46-P more weakly than HPr-Ser46-P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser46-P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser46-P)-DNA complex. The structure reveals that Crh-Ser46-P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser46-P. The reduced affinity of Crh-Ser46-P for CcpA as compared with that of HPr-Ser46 P is explained by weaker Crh-Ser46-P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix alpha 2 of the second contact region (contact region II) of Crh-Ser46-P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA.

Highlights

catabolite control protein A (CcpA) is a member of the LacI-GalR family of transcription regulators [14]
Like histidine-containing protein (HPr), Crh does contain the Ser46 residue that is phosphorylated by the enzyme HPr kinase/phosphorylase, and Crh has been demonstrated to function in carbon catabolite repression/regulation (CCR) [28, 31]
Gested that Crh residue Gln15 would be able to interact with CcpA residue Asp296 in a manner similar to the interaction observed between His15 and Asp296 in the CcpA-(HPr-Ser46-P)-cre structure [27]

Summary

Introduction

CcpA is a member of the LacI-GalR family of transcription regulators [14]. LacI-GalR proteins contain a 60-residue N-terminal DNA binding domain that connects to a larger C-terminal domain. Examination of the CcpA-(HPr-Ser46-P)-cre complex structure shows that, with the exception of two residues (HPr 3 Crh: H15Q, T20A), the expected CcpA binding interfaces of the two proteins should be identical. Our structure, obtained under high protein concentrations (200 ␮M) clearly reveals that the monomer of Crh-Ser46-P functions as a corepressor for CcpA, a finding consistent with recent studies examining CcpA binding to Crh-Ser46-P and HPr-Ser-46-P by surface plasmon resonance [34].

Results

Conclusion