Hierarchical cross-linking of a bacterial spore coat Hub protein

Abstract

Hub proteins are highly connected nodes in protein-protein interaction networks and are often intrinsically disordered proteins (IDPs) or contain intrinsically disordered regions. In Bacillus subtilis, the morphogenesis of the spore surface is orchestrated by a set of so-called morphogenetic proteins that guide the assembly of distinct layers. Formation of the inner coat is directed by SafAFL and its shorter isoform, C30. Both are expressed early in sporulation under the control of {sigma}E and localize at the interface between the developing inner coat and the underlying cortex peptidoglycan. From this site, they act as organizational hubs, recruiting client proteins essential for coat maturation. Among these is Tgl, a transglutaminase synthesized later in development following activation of {sigma}K after engulfment completion. We show that the C30 domain exhibits IDP-like features yet self-assembles into >1200 kDa complexes stabilized by disulfide bonds and that these bonds are required for subsequent proper Tgl-mediated "spotwelding" cross-linking. Small-angle X-ray scattering (SAXS) and photobleaching show that Tgl immobilizes but does not drastically alter these assemblies. These findings support a hierarchical, biphasic model for inner coat assembly: initial self-assembly and disulfide stabilization, followed by Tgl-mediated cross-linking and structural stabilization. According to this model, the forms of SafAFL/C30 that dominate the two stages recruit different client proteins in register with the course of morphogenesis.