Insight into the autoproteolysis mechanism of the RsgI9 anti-σ factor from Clostridium thermocellum.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/ß/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing.

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0 Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit ~160 Å from the cell surface. The CRE and proline-rich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.

Antimicrobial Peptide-Polymer Conjugates with High Activity: Influence of Polymer Molecular Weight and Peptide Sequence on Antimicrobial Activity, Proteolysis, and Biocompatibility.

We report the synthesis, characterization, activity, and biocompatibility of a novel series of antimicrobial peptide-polymer conjugates. Using parent peptide aurein 2.2, we designed a peptide array (∼100 peptides) with single and multiple W and R mutations and identified antimicrobial peptides (AMPs) with potent activity against Staphylococcus aureus (S. aureus). These novel AMPs were conjugated to hyperbranched polyglycerols (HPGs) of different molecular weights and number of peptides to improve their antimicrobial activity and toxicity. The cell and blood compatibility studies of these conjugates demonstrated better properties than those of the AMP alone. However, conjugates showed lower antimicrobial activity in comparison to that of peptides, as determined from minimal inhibition concentrations (MICs) against S. aureus, but considerably better than that of the available polymer-AMP conjugates in the literature. In addition to measuring MICs and characterizing the biocompatibility, circular dichroism spectroscopy was used to investigate the interaction of the novel conjugates with model bacterial biomembranes. Moreover, the novel conjugates were exposed to trypsin to evaluate their stability. It was found that the conjugates resist proteolysis in comparison with unprotected peptides. The peptide conjugates were active in serum and whole blood. Overall, the results show that combining a highly active AMP and low-molecular-weight HPG yields bioconjugates with excellent biocompatibility, MICs below 100 µg/mL, and proteolytic stability, which could potentially improve its utility for in vivo applications.