Small angle X-ray scattering analysis of Clostridium thermocellum cellulosome N-terminal complexes reveals a highly dynamic structure

Abstract

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.

FIGURE 1.

Schematic representation of the cellulosome from C. thermocellum. The cellulosome is composed of three types of proteins: 1) a cell surface-anchoring protein, 2) the CipA scaffoldin protein, and 3) enzyme subunits. Cell-surface proteins are composed of CohII modules (white C) and surface-like homology (SLH) domains, which interact with the cell surface. CipA is composed of a DocII module (white D), an X module (X), a family 3a cellulose-specific binding module (the CBM), and nine CohI modules (black C_1–9) enumerated from the N terminus, which is on the left side in this schematic. Enzyme subunits contain a DocI module (black D) and a catalytic module (black E). The (CohI₁-CohI₂)·2Cel8A and (CohI₂-CBM-CohI₃)·2Cel8A complexes and where they map in the full-length cellulosome are shown in the solid and dashed boxes, respectively.

FIGURE 2.

SAXS data and analysis of the (CohI₁-CohI₂)·2Cel8A (left panels) and (CohI₂-CBM-CohI₃)·2Cel8A (right panels) complexes. A, SAXS data (black) and minimal ensemble search fit (red). B, Guinier plots shown for q*R_g < 1.3. C, pair distribution function. The results indicate that both complexes are monomeric and elongated in solution.

FIGURE 3.

Best fit ab initio envelopes for the (CohI₁-CohI₂)·2Cel8A complex. Envelopes generated by DAMMIN (A) and GASBOR (B) are displayed in translucent light gray. CipA CohI and CBMs are colored yellow and dark gray, respectively. Both the DocI and catalytic modules of the Cel8A enzymes are shown in red.

FIGURE 4.

Best fit ab initio envelopes for the (CohI₂-CBM-CohI₃)·2Cel8A complex. Envelopes generated by DAMMIN (A) and GASBOR (B) are displayed in translucent light gray. CipA CohI and CBMs are colored yellow and dark gray, respectively. Both the DocI and catalytic modules of the Cel8A enzymes are shown in red.

FIGURE 5.

Rigid body models of the (CohI₁-CohI₂)·2Cel8A (A) and (CohI₂-CBM-CohI₃)·2Cel8A (B) complexes. The protein structures are colored as described in the legend to Fig. 3.

FIGURE 6.

The (CohI₁-CohI₂)·2Cel8A minimal ensemble. The protein structures are colored as described in the legend to Fig. 3. All eight conformers are displayed. The percentages represent the fraction of a given conformer in the minimal ensemble. A highly dynamic complex with no conformational preferences is revealed.

FIGURE 7.

The (CohI₂-CBM-CohI₃)·2Cel8A minimal ensemble. The protein structures are colored as described in the legend to Fig. 3. All five conformers are displayed. The percentages represent the fraction of a given conformer in the minimal ensemble. Consistent with the (CohI₁-CohI₂)·2Cel8A complex, the (CohI₂-CBM-CohI₃)·2Cel8A complex is also highly dynamic with no conformational preferences.