Biology subjects

Ruotolo, B.

Publications and source records attributed to Ruotolo, B..

2 recordsLinked to original sources

bioRxiv2026

Structural analyses of a heterodimeric SusCD complex captures intermediate states of maltooligosaccharide transport

Bacteroides are abundant gut bacteria with diverse polysaccharide metabolizing capabilities encoded by co-transcribed polysaccharide utilization loci (PULs). The starch utilization system (Sus) is required for starch and a-glucan metabolism in Bacteroides thetaiotaomicron and has served as a model for PUL studies. However, the mechanism of maltooligosaccharide (MOS) transport and the architecture of the SusCD complex remain incompletely understood. Here, we used cryo-electron microscopy to capture multiple transport-relevant conformations of the SusD lipoprotein and the TonB-dependent transporter SusC in both unliganded and ligand-bound states. In the absence of ligand, SusCD adopts three conformations including an open state in which SusD is displaced from SusC, a state containing the SusC plug, and a state lacking density for the SusC plug. Three-dimensional variability analysis of the structure where SusD is displaced from SusC reveals continuous SusD mobility coupled to movement of the SusC N-terminus. The maltoheptaose-bound SusCD structure reveals two binding sites, one at the SusD-SusC interface and a second within SusC, indicating how MOS stabilizes a closed SusCD assembly. Notably, all structures show that SusCD is a heterodimer, unlike other Sus-like transporters that assemble into SusCD heterotetramers. Native mass spectrometry confirms this heterodimeric stoichiometry, and comparisons with other SusCD-like complexes suggest a structural basis for this distinct assembly of SusCD. Together, these data define substrate binding and intermediate transport states in a canonical PUL system and reveal architectural divergence among Sus-like carbohydrate transport complexes in Bacteroides.

microbiology↗

bioRxiv2025

The bacterial chaperone CsgC inhibits functional amyloid CsgA formation by promoting the intrinsically disordered pre-nuclear state

E. coli assembles a functional amyloid called curli during biofilm formation. The major curlin subunit is the CsgA protein, which adopts a beta-sheet rich fold upon fibrillization. The chaperone-like protein CsgC inhibits CsgA amyloid formation. CsgA undergoes a 3-stage aggregation process: an initial lag phase where beta-rich nuclei form, an exponential elongation phase, and a plateau phase. It is currently not known if CsgC inhibits amyloid formation by inhibiting formation of a pre-fibril nucleus, or if CsgC inhibits a later stage of amyloid formation by blocking monomer addition. Here, CsgC homologs from C. youngae, C. davisae, and H. alvei were purified and characterized for their ability to interrogate CsgA amyloid formation. Each of the CsgC homologs prolonged the lag phase of E. coli CsgA amyloid formation similar to E. coli CsgC. Additionally, we found E. coli CsgC interacted transiently and weakly with a monomeric, pre-nucleus species of CsgA which delayed amyloid formation. A transient CsgC-CsgA heterodimer was observed using ion mobility-mass spectrometry. When CsgC was added to actively polymerizing CsgA, exponential growth commonly associated with nucleation-dependent amyloid formation was lost. Adding preformed CsgA seeds did not rescue exponential growth, indicating that CsgC also has inhibitory activity during fibril elongation. Indeed, CsgC interacted strongly with CsgA fibers, suggesting the interaction between CsgC and CsgA fibers can slow new fiber growth. CsgC displays unique inhibitory activity at multiple stages of amyloid formation. CsgC acts as an energy-independent chaperone that transiently interacts with prefibrillar CsgA and an amyloid fiber.

biochemistry↗