Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species

Abstract

Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, tāpirins, located proximate to the type IV pilus locus. Previously, the C-terminal domain of a tāpirin (Calkro_0844) from Caldicellulosiruptor kronotskyensis was shown to be 40 structurally unique and have a cellulose binding affinity akin to family 3 carbohydrate binding modules (CBM3). Here, full-length and C-terminal versions of tāpirins from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis (Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five tāpirins bound to 45 microcrystalline cellulose, switchgrass, poplar, filter paper, but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844 and NA10_0869 (from strongly cellulolytic species), perhaps to associate closely with biomass to capture glucans released from lignocellulose by cellulases 50 produced in Caldicellulosiruptor communities. Three-dimensional structures of the C-terminal binding regions of Calhy_0908 and Calkr_0826 were closely related to Calkro_0844, despite the fact that their amino acid sequence identities compared to Calkro_0844 were only 16% and 36%, respectively. Unlike the parent strain, C. bescii mutants lacking the tāpirin genes did not bind to cellulose following short-term incubation, reinforcing the significance of these proteins in cell 55 association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, tāpirins likely help cells scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species. 65binding proteins, called tāpirins, are involved in the way Caldicellulosiruptor species interact with microcrystalline cellulose and here additional information about the diversity of these proteins across the genus is provided, including three dimensional structural comparisons. and 15% to 24% w/v PEG 3350 (best crystals appeared in 0.015 M zinc acetate and PEG 3350 concentration of 17-18%). The protein solutions contained 7.5 mg/mL of protein, 20 mM Tris pH 7.5, 100 mM NaCl and 2% of the Hampton Research Tacsimate pH 7 mix.All crystals were soaked in well solution with PEG 3350 increased to 25% along with 5-10% ethylene glycol added for the cryo protection. For the purpose of structure determination, an 355 iodine derivative was obtained for the Calhy_0908C by quick soaking the crystals in the cryoprotectant described above with 0.1 M potassium iodide added. Crystallography data collection and processing. The crystals of Calkr_0826C andCalhy_0908C were flash frozen in a nitrogen gas stream at 100 K before home source data collection using an in-house Bruker X8 MicroStar X-Ray generator with Helios mirr...

Caldicellulosiruptor besciiregulates pilus expression in response to the polysaccharide, xylan

“…S4A). As expected, the tāpirin protein was capable of adsorbing to microcrystalline cellulose at 25˚C, as 210 was previously observed (19,22), however a denser band was observed after incubation at 75˚C ( Fig. S4B), similar to our observations with rCbPilA.…”

“…8). In addition, CbPilA may function along with the tāpirins (19,22) to promote cell adherence to cellulose and we propose a model where cell adhesion to cellulose involves both proteins (Fig. 8).…”

“…Structurally unique proteins called tāpirins are another mechanism by which strongly to weakly cellulolytic Caldicellulosiruptor species attach to cellulose from plant biomass (19,22). Genes encoding for tāpirins are located directly downstream of the T4P locus in all cellulolytic Caldicellulosiruptor (10,19,22), however it remains to be determined if they interact with the T4P. 70…”