Mahoney Brendan J, Takayesu Allen, Zhou Anqi, Cascio Duilio, Clubb Robert T
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA.
UCLA-DOE Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California, USA.
Proteins. 2022 Jul;90(7):1457-1467. doi: 10.1002/prot.26326. Epub 2022 Feb 28.
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.
热纤梭菌正作为一种微生物平台积极开展研究，用于从可再生植物生物质中生产生物燃料和化学品。这种细菌的一个吸引人的特性是它能够利用表面展示的纤维小体高效降解木质纤维素，纤维小体是容纳不同类型纤维素酶的大型多蛋白复合物。热纤梭菌利用9种膜嵌入抗σ因子（RsgI1 - 9）来调整其纤维小体的酶组成，这些抗σ因子被认为能够感知不同类型的细胞外碳水化合物，然后通过细胞质σ因子引发不同的基因表达程序。在这里，我们表明RsgI9抗σ因子通过一个C端双结构域单元与纤维素相互作用。一个2.0 Å的晶体结构显示，该单元由S1C肽酶和类NTF2蛋白结构域构成，其中含有一个潜在的纤维素结合位点。完整胞外结构域的小角X射线散射实验表明，它呈现出双叶状的细长构象。在该结构中，一个保守的RsgI胞外（CRE）结构域通过富含脯氨酸的连接子与双结构域相连，预计该连接子会将碳水化合物结合单元从细胞表面伸出约160 Å。CRE和富含脯氨酸的元件在其他几种热纤梭菌抗σ因子中是保守的，这表明它们也会形成能够感知碳水化合物的延伸结构。
