P1 ParB domain structure includes two independent multimerization domains.
ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.
Surtees
J A
JA
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Funnell
B E
BE
D000595:Amino Acid Sequence; D017101:Bacteriophage P1 / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D019281:Dimerization; D010450:Endopeptidases; D004926:Escherichia coli / Q000821:virology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011533:Proviruses / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017421:Sequence Analysis; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; C121785:ParB protein, Enterobacteria phage P1; D010446:Peptide Fragments; D014764:Viral Proteins; D010450:Endopeptidases