Solution structure and dynamics of crh, the bacillus subtilis catabolite repression HPr

Abstract

The solution structure and dynamics of the Bacillus subtilis HPr-like protein, Crh, have been investigated using NMR spectroscopy. Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr. Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be monomeric. Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allowing the identification of the Crh dimer interface from chemical-shift mapping. The conformation of Crh was determined to a precision of 0.46(±0.06) Å for the backbone atoms, and 1.01(±0.08) Å for the heavy atoms. The monomer structure is similar to that of known HPr 2.67(±0.22) Å (C α rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in β-sheet pairing of the N-terminal strand with the β4 strand. This shift results in a shortening of the surface loop present in HPr and consequently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two proteins. A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr. This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA. 15N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site.