Combining chromatography coupled SAXS and AIpredicted structures to dissect the mechanism of ParB1parS1 partition assembly formation

Coupling of solution SAXS and AI-predicted structures can be a powerful strategy for delineating subtle conformational changes and self-association in protein switches. ParB, which is a condensate-forming DNA clamp that aids in bacterial chromosomal origin segregation, undergoes CTP-induced conformational switching to enable DNA sliding. The nature of parS DNA-induced conformational change in full-length ParB, and the structural features that govern self-association of ParB for partition assembly condensate formation, remains sparsely understood. We combined chromatography-coupled SAXS, rigid domains obtained from Alphafold model of ParB1 from Vibrio cholerae, and synthetic SAXS data describing known domain interfaces, to build integrative models of conformational states of full-length ParB1. These integrative models revealed how parS1 DNA loading primed ParB1 for clamping and sliding. The CTPase domains in ParB1 were moved nearer upon DNA loading to facilitate clamping, and a lumen lined with a weak DNA binding site was formed below parS1 binding site for capturing the sliding DNA. Furthermore, we showed that an N-terminal segment of ParB1 undergoes concentration-dependent oligomerization. An intrinsically disordered linker joining this oligomerization-prone N-terminal segment and the C-terminal domain curbs self-association of full-length ParB1, which is likely relevant for ParB1-mediated higher order partition assembly formation. To summarize, SAXS and Alphafold were effectively combined to provide unique insights into context-specific domain rearrangements and self-association in ParB1 for the mechanistic understanding of partition assembly formation.