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, called ta pirins, located proximate to the type IV pilus locus. The C-terminal domain of Caldicellulosiruptor kronotskyensis ta pirin 0844 (Calkro_0844) is structurally unique and has a cellulose binding affinity akin to that seen with family 3 carbohydrate binding modules (CBM3s). Here, full-length and C-terminal versions of ta pirins 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 ta pirins bound to microcrystalline cellulose, switchgrass, poplar, and 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 this relates to their specific needs to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Calkro_0844 and NA10_0869 share a higher degree of amino acid sequence identity (Ͼ80% identity) with each other than either does with Athe_1870 (ϳ50%). The levels of amino acid sequence identity of Calhy_0908 and Calkr_0826 to Calkro_0844 were only 16% and 36%, respectively, although the three-dimensional structures of their C-terminal binding regions were closely related. Unlike the parent strain, C. bescii mutants lacking the ta pirin genes did not bind to cellulose following short-term incubation, suggesting a role in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, ta pirins likely help scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species. IMPORTANCE The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called ta pirins, are involved in the way that Caldicellulosiruptor species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…Genes encoding for uncharacterized hypothetical proteins, later identified as tāpirins, are located directly upstream of the GDL, and were initially demonstrated to have micromolar affinity for microcrystalline cellulose along with an unusual β-helix shape [36]. Two additional tāpirin structures were recently solved from divergent tāpirins produced by weakly cellulolytic species (C. hydrothermalis and C. kristjanssonii) that share a similar shape to the highly cellulolytic tāpirin proteins [37]. Furthermore, biophysical analysis of tāpirins from weakly cellulolytic species indicates that these tāpirins bind to more sites on cellulose, likely aided by a longer predicted binding pocket in their structure [36,37], and grant a competitive advantage to adhere to plant biomass in their environment.…”

“…Two additional tāpirin structures were recently solved from divergent tāpirins produced by weakly cellulolytic species (C. hydrothermalis and C. kristjanssonii) that share a similar shape to the highly cellulolytic tāpirin proteins [37]. Furthermore, biophysical analysis of tāpirins from weakly cellulolytic species indicates that these tāpirins bind to more sites on cellulose, likely aided by a longer predicted binding pocket in their structure [36,37], and grant a competitive advantage to adhere to plant biomass in their environment. Not all strongly cellulolytic members of the genus Caldicellulosiruptor use classical tāpirins to adhere to cellulose, as atypical tāpirin proteins similar to those encoded for by C. owensensis and C. acetigenus were identified in the genome of C. changbaiensis [22].…”

Genomic and physiological analyses reveal that extremely thermophilicCaldicellulosiruptor changbaiensisdeploys unique cellulose attachment mechanisms

“…This observed sequence divergence between the atypical tāpirins from strongly and weakly cellulolytic species is similar to the tāpirin encoded for by C. hydrothermalis which shares little amino acid sequence homology with classical tāpirins, but shares a similar tertiary structure, and is capable of occupying more sites on crystalline cellulose in comparison to classical tāpirins [37]. Production of tāpirins with an affinity to cellulose likely plays a role in the ability of weakly cellulolytic members of the genus to adhere to cellulose and benefit from the cellooligosaccharides released by the action of cellulases [60].…”

“…1). Overall, there are 120 unique protein clusters identified in the C. changbaiensis genome when compared to the prior Caldicellulosiruptor pangenome [37], 75 of which were annotated as hypothetical proteins. Further transcriptomic and proteomic studies may aid in the identification of the function of these unique hypothetical proteins.…”

“…The availability of genome sequences has precipitated deeper insights into the genus Caldicellulosiruptor , including comparative studies which have identified biomarkers for plant biomass deconstruction [6,5,23], novel insertion elements [15], genetic tractability [11], diverse mechanisms involved in biomass solubilization [66,37], unique cellulose adhesins (tāpirins) [5,37] and the identification of new combinations of catalytic domains [5,36,23]. Perhaps owing to the unique thermal environments that this genus inhabits, their genomes appear to be dynamic, as the first described Caldicellulosiruptor pangenome was predicted to be open [5], and remained open after the addition of five additional genome sequences [36].…”