Novel clostridial cell-surface hemicellulose-binding CBM3 proteins

Abstract

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Å resolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

Keywords: CBM; Clostridium clariflavum; Clostridium thermocellum; calcium binding; cellulosome; crystal structure.

Figure 1

Overview of the CtCBM3-0271 molecule. (a) ClustalW-generated amino-acid sequence alignment of the orthologous CBM3-containing open reading frames (ORFs) from C. thermocellum and C. clariflavum. The proposed transmembrane regions are underlined. The CBM regions are denoted in rectangles. (b) Genomic organization of the region in proximity to Cthe_0271 (cyan) in C. thermocellum ATCC 27405. Genes are depicted as arrows. Gene nomenclature is given below. Names of proteins related to carbohydrate utilization (annotations/predicted functions) are indicated at the top. Cthe_0267, alternative anti-sigma factor 2; Cthe_0268, RNA polymerase (alternative) sigma factor 2; Cthe_0269, family 8 endoglucanase; Cthe_0270, family 18 glycoside hydrolase (chitinase); Cthe_0271, the type 3 CBM-containing protein investigated in this work; Cthe_0272, serine-type d-alanyl-d-alanine carboxypeptidase; Cthe_0273, diguanylate cyclase; Cthe_0274, family 9 glycoside hydrolase; Cthe_0275: family 36 glycosyl transferase. The figure was created using Gene Graphics (https://katlabs.cc/genegraphics/app). (c) Schematic model of the CtCBM3-0271 and CcCBM3-1192 proteins with the CBM3 at the C-terminus, a linker, an unknown domain, a transmembrane helix (TMH) and a short intracellular segment at the N-terminus. The residue numbers correspond to the CtCBM3-0271 sequence.

Figure 2

Binding properties of CtCBM3-0271 and CcCBM3-1192. (a) Binding to insoluble polysaccharides. The desired recombinant protein was incubated with the indicated insoluble polysaccharides, centrifuged and the bound (+) and unbound (−) fractions were analyzed by SDS–PAGE. Both CBM3s show binding of xylan and chitin, but no binding of cellulose or lichenan. (b) Binding of CtCBM3-0271 and CcCBM3-1192 to oat spelt xylan at increasing concentrations of NaCl as indicated. Xylan binding by both CBM3s is affected by increased NaCl concentrations, resulting in decreased CBM3 binding towards xylan.

Figure 3

The 3D structures of CtCBM3-0271 and CcCBM3-1192 with that of CtCipA CBM3a for comparison. Cartoon representations of the secondary-structural elements are shown with numbered β-strands. The N- and C-termini are indicated and the calcium ions in CtCBM3-0271 and CtCipA CBM3a are shown as spheres. CcCBM3-1192 does not contain a calcium ion.

Figure 4

Potential ‘linear-strip’ cellulose-binding regions in selected members of the CBM3 family. Surface representations are shown of the designated CBM3 molecules, with an emphasis on plausible residues that could play a role in carbohydrate binding. The residues forming the cellulose-binding strip are highlighted in yellow, identified and numbered. The topographic obstacle on the cellulose-binding surface of CBM3-0267 is colored in a darker green tint.

Figure 5

The calcium-binding regions of CBM3s. (a) Superposition of the calcium-binding loop regions of CtCBM3-0271 (cyan) and CcCBM3-1192 (magenta). The amino-acid residues contributing to the coordination are located on L3,4 and L7,8. Calcium-binding residues of CtCBM-0271 are shown as sticks and labeled. The loops of CcCBM3-1192 show a broader configuration compared with those of CtCBM3-0271, probably due to the lack of calcium, which thus does not constrict them into a tighter arrangement. (b) Stick representations of the calcium-binding residues of CtCBM3-0271 (left) and CcCBM3-1192 (right).