Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force...

Protein interactions typically involve a single, well-defined binding conformation. However, some systems, such as cellulosomal cohesin-dockerin complexes, are hypothesized to form through multiple distinct binding modes. Prior studies have established that these complexes are essential for the function of cellulosomes, which are multi-enzyme structures in bacteria. Despite this, the exact structural and mechanical details of dual binding modes remain unclear. Bulk biochemical methods have failed to distinguish between the modes due to their similar thermodynamic profiles. This gap motivated the use of single-molecule techniques to resolve conformational differences. The need for high-resolution mechanical data has led to the adoption of force spectroscopy approaches. No prior work had resolved the mechanical signatures of dual binding modes in Coh-Doc interactions. This uncertainty drove the development of a mutagenesis and force spectroscopy strategy.

This study aimed to investigate whether cellulosomal cohesin-dockerin complexes exhibit dual binding conformations. The researchers sought to overcome the limitations of bulk methods by using single-molecule force spectroscopy. They hypothesized that distinct mechanical signatures could reveal the existence of multiple binding modes. The specific problem addressed was the inability of traditional methods to detect conformational heterogeneity in Coh-Doc interactions. The motivation stemmed from the need to understand how these complexes assemble and function in vivo. The study focused on the Clostridium thermocellum exocellulase Cel48S system. By combining mutagenesis with force spectroscopy, the team aimed to isolate and characterize each binding mode. The ultimate goal was to determine the relative populations of the two binding conformations under native conditions.

The researchers used site-directed mutagenesis to create dockerin variants that selectively disrupted one of the two proposed binding modes. Single-molecule force spectroscopy (SMFS) was performed using an atomic force microscope (AFM) cantilever to measure unbinding forces. The AFM setup allowed for controlled application of mechanical force to dissociate the Coh-Doc complexes. Each mutation was designed to target a specific conformational pathway. The unfolding patterns of the mutated Docs were compared to the wild-type protein. Bulk biochemical assays were also conducted to confirm the absence of global structural changes. The mechanical response of each mutant was analyzed to determine if it corresponded to a specific binding mode. This approach enabled the discrimination of binding modes that were indistinguishable in bulk measurements.

The study found that wild-type Docs from Clostridium thermocellum Cel48S exhibited two distinct mechanical signatures during unbinding. Each binding mode was associated with a unique force threshold and unfolding pattern. The mutated Docs selectively eliminated one of the two binding modes, confirming their existence. The first mode required a lower force for dissociation compared to the second. The mechanical differences were not detectable in bulk assays due to their similar thermodynamic profiles. The force spectroscopy data revealed that both binding modes were populated with nearly equal probability. This finding suggests that the dual binding mechanism is a native feature of the Coh-Doc interaction. The results provide direct evidence for conformational heterogeneity in these complexes.

The authors conclude that the wild-type dockerin from Clostridium thermocellum Cel48S exists in two distinct binding conformations under native conditions. The use of single-molecule force spectroscopy allowed the discrimination of these modes, which were indistinguishable in bulk measurements. The study confirms that the dual binding mechanism is a functional feature of the Coh-Doc interaction. The mechanical properties of each mode suggest different roles in complex stability and assembly. The findings support the hypothesis that multiple conformations contribute to the robustness of cellulosome formation. The approach used here can be applied to other Coh-Doc systems with predicted dual binding modes. The results open new avenues for studying the mechanical basis of multi-enzyme complex assembly. The study provides a framework for future investigations into the structural dynamics of similar protein interactions.