A mutational study of Cnu reveals attractive forces between Cnu and H-NS

Abstract

Cnu is a small 71-amino acid protein that complexes with H-NS and binds to a specific sequence in the replication origin of the E. coli chromosome. To understand the mechanism of interaction between Cnu and H-NS, we used bacterial genetics to select and analyze Cnu variants that cannot complex with H-NS. Out of 2,000 colonies, 40 Cnu variants were identified. Most variants (82.5%) had a single mutation, but a few variants (17.5%) had double amino acid changes. An in vitro assay was used to identify Cnu variants that were truly defective in H-NS binding. The changes in these defective variants occurred exclusively at charged amino acids (Asp, Glu, or Lys) on the surface of the protein. We propose that the attractive force that governs the Cnu-H-NS interaction is an ionic bond, unlike the hydrophobic interaction that is the major attractive force in most proteins.

Fig. 1.

The plasmid map of pCnu and pOri14 (A). pCnu encodes the Cnu protein that binds as a Cnu-H-NS complex to the Ori-14 sequence in the operator region of pOri14. This binding activity makes the host cell (HB101) resistant to streptomycin. The DNA sequence of the oriC region in E. coli (B). The 13-mer regions are where DNA opening occurs during the initiation of chromosomal DNA replication. R and M regions are where DnaA protein binds. The Cnu-H-NS binding site (cnb) identified by (Kim et al., 2005) is overlined and the refined binding site (Ori-14 from this study) within cnb is boxed. The Growth Ratio (GR) of HB101 harboring the indicated plasmids are presented (C). Higher GRs correspond to better binding of the Cnu-H-NS complex to the Ori-14 sequence in vivo.

Fig. 2.

Tricine-SDS-polyacrylamide protein gels indicate that H-NS is co-eluted with the His-tagged Cnu from the nickel affinity column (A). The same protein gels from some of the Cnu variants (B) showing that H-NS is not co-eluted with the corresponding Cnu variant. F10I and M39C Cnu variants are not detected in the protein gel. Some variants, such as S15P and Q70H, are co-eluted with H-NS (C). Cnu^* shows that H-NS is not co-eluted when the His-tagged Cnu is prepared from hns-deleted BL21(DE3). M indicates a protein molecular marker. Nine micrograms of protein were loaded in each lane.

Fig. 3.

A schematic presentation of the side chains of the Cnu variants belonging to the internal group. The corresponding side chains are shown in white. Alpha helices (H1, H2, and H3) are indicated by a cylinder with arrow head that indicates the direction of the C-terminus. This 3D structure of Cnu was drawn using Cn3D (Wang et al., 2000) based on Protein data bank ID 2JQT.

Fig. 4.

A schematic presentation of the side chains of the Cnu variants belonging to the external group. The corresponding side chains are shown in white.

Fig. 5.

A schematic presentation of the side chains of the Cnu variants belonging to the loop group. The corresponding side chains are shown in white.