Pina, A. S. | Search bioRxiv

bioRxiv2026

Hierarchical cross-linking of a bacterial spore coat Hub protein

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.

microbiology↗

bioRxiv2024

Investigating Amino acid Enrichments and Patterns in Phase-Separating Proteins: Understanding Biases in Liquid-Liquid Phase Separation

Liquid-Liquid Phase Separation (LLPS) forms membraneless organelles, enhancing biochemical processes. The stickers-and-spacers model explains LLPS but is mainly validated in Prion-like RNA Binding Proteins. We explore peptide motifs in LLPS in broader protein contexts. We developed a computational approach for motif discovery, implemented in 178 Phase-Separating Proteins (PhSePs), complemented by the FuzDrop and CIDER servers, which identified droplet-promoting regions (DPRs) and examined disorder-related characteristics. Our database of PhSePs was analyzed against proteins with low propensity for LLPS. This comparative analysis revealed 129 enriched peptide motifs with folds higher than 0.2, consisting of 3 to 6 residues, with tetrapeptides being the most prevalent. Key features of the enriched motifs included Gly-rich sequences punctuated with aromatic, charged, and polar residues, as well as homopeptide repeats (e.g., GGDR, SRGG, YGGG, QQQQ, PPPP). Analysis of motif presence, frequency, and co-occurrence revealed widely distributed motifs across different DPRs, identified motifs with significant repetitive patterns, and highlighted motif trios that are more likely to co-occur within a sequence. By harvesting this analysis, we developed a data-driven approach for minimalistic peptide design with LLPS propensity, further using the CIDER server for peptide characterization and peptide design refinement. We designed 8 peptides with various motif combinations and amino acid distributions, which were experimentally validated to undergo LLPS, exhibiting liquid-like behavior with diverse molecular mobility patterns and droplet dynamics. Our approach bridges a non-biased computational approach with experimental validation, offering insights into sequence determinants of phase separation, with the potential for designing minimalistic synthetic condensates with tailored properties.

biochemistry↗