Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, situating it among the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components can help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass.

Here we employed single-molecule force spectroscopy with an atomic force microscope (AFM) and steered molecular dynamics (SMD) simulations to reveal force propagation pathways through a mechanically ultrastable multi-domain cellulosome protein complex. We demonstrate a new combination of network-based correlation analysis supported by AFM directional pulling experiments, which allowed us to visualize stiff and soft paths through the protein complex along which force was transmitted. The results implicate specific force-propagation architectures non-parallel to the pulling axis that are advantageous for achieving high dissociation forces.

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Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from Ruminococcus flavefaciens called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 105 pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics.

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicteddual binding modes across multiple bacterial species, our approach opens up newpossibilities for understanding assembly and catalytic properties of a broadrange of multi-enzyme complexes.DOI: http://dx.doi.org/10.7554/eLife.10319.001