Distinctive ligand‐binding specificities of tandem PA14 biomass‐sensory elements from Clostridium thermocellum and Clostridium clariflavum

Abstract

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi‐enzyme cellulosome complex, certain clostridia contain alternative sigma I (σI) factors that have cognate membrane‐associated anti‐σI factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure‐function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14‐superfamily motifs. The X‐ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σI4‐dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σI3‐dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation‐related genes. Structural similarity between clostridial PA14 dyads to PA14‐containing proteins in yeast helped identify another crucial signature element: the calcium‐binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14A) is dominant in directing the binding to the ligand in both bacteria. The two X‐ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

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

“…The RsgI1, RsgI2, RsgI3, RsgI4, and RsgI6 anti‐σ factors in C. thermocellum bind to carbohydrates and are thought to serve as biomass sensors 20–22 . We therefore probed whether RsgI9 could interact with cellulose using an established pull‐down assay 20,22 .…”

“…Binding to the microcrystalline cellulose substrate (Avicel, Sigma‐Aldrich) was qualitatively assayed via SDS‐PAGE pull‐down assays modified from previous protocols 20,22 . Recombinant protein and control samples were prepared using 50 μg of protein in 200 μl of lysis buffer (50 mM Tris–HCl, 300 mM NaCl, pH 8.0) and mixed with 15 μg of microcrystalline cellulose (Avicel).…”

“…However, our understanding of how these regulatory systems sense carbohydrates and alter gene expression remains limited. In vitro binding studies have experimentally identified carbohydrates that interact with the RsgI1, RsgI2, RsgI3, RsgI4, and RsgI6 anti‐σ factors, but the specific genes whose expression is ultimately regulated by these binding events is generally not known 18,20–22 . This is because at present only partial regulons have only been identified for SigI1, SigI3, and SigI6 using bioinformatics analyses and verified experimentally 17 .…”

“…In vitro binding studies have experimentally identified carbohydrates that interact with the RsgI1, RsgI2, RsgI3, RsgI4, and RsgI6 anti-σ factors, but the specific genes whose expression is ultimately regulated by these binding events is generally not known. 18,[20][21][22] This is because at present only partial regulons have only been identified for SigI1, SigI3, and SigI6 using bioinformatics analyses and verified experimentally. 17 Finally, the molecular mechanism through which extracellular carbohydrate binding to each anti-σ factor triggers gene expression by releasing its cognate ECF σ factor remains to be determined.…”

Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253

“…2) and active on glucosylated macrolides 50 . The PA14 domain in other carbohydrate-active proteins, including the closely related KmBglI from Kluyveromyces marxianus, have been associated with oligosaccharide binding 32,51,52 . However, no substrate interaction possibility for the PA14 domain was found, neither in the model of RmBgl3B nor in the structure of DesR.…”