Crystal structure and centromere binding of the plasmid segregation protein ParB from pCXC100.
Plasmid pCXC100 from the Gram-positive bacterium Leifsonia xyli subsp. cynodontis uses a type Ib partition system that includes a centromere region, a Walker-type ATPase ParA and a centromere-binding protein ParB for stable segregation. However, ParB shows no detectable sequence homology to any DNA-binding motif. Here, we study the ParB centromere interaction by structural and biochemical approaches. The crystal structure of the C-terminal DNA-binding domain of ParB at 1.4 Å resolution reveals a dimeric ribbon-helix-helix (RHH) motif, supporting the prevalence of RHH motif in centromere binding. Using hydroxyl radical footprinting and quantitative binding assays, we show that the centromere core comprises nine uninterrupted 9-nt direct repeats that can be successively bound by ParB dimers in a cooperative manner. However, the interaction of ParB with a single subsite requires 18 base pairs covering one immediate repeat as well as two halves of flanking repeats. Through mutagenesis, sequence specificity was determined for each position of an 18-bp subsite. These data suggest an unique centromere recognition mechanism by which the repeat sequence is jointly specified by adjacent ParB dimers bound to an overlapped region.
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China.
Lin
L
Huang
Ping
P
Yin
Xing
X
Zhu
Yi
Y
Zhang
Keqiong
K
Ye
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002503:Centromere / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C052883:chromosome partition proteins, bacterial