Mechanisms of Nanonewton Mechanostability in a Protein Complex Revealed by Molecular Dynamics Simulations and Single-Molecule Force Spectroscopy

Abstract

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.

High Force Catch Bond Mechanism of Bacterial Adhesion in the Human Gut

“…1a, purple). This newly reported complex is homologous to previously reported complexes from R. flavefaciens (Rf) [35][36][37] . Full amino acid sequences are given in the Supplementary Information.…”

“…Therefore, at high loading rates, far fewer complexes reach sufficiently high forces to unfold XMod prior to complex rupture, thus prohibiting the system from entering P2. This behavior is unique to this particular XMod-Doc:Coh system and was not observed in other Dockerin-Cohesin systems reported thus far 35,36 .…”

“…From the 5 strongest models for each binding mode, we performed 200 production SMD simulation replicas, using a similar protocol as previously described 26,35 . The simulations reveal that the dissociation of XMod-Doc:Coh occurs at clearly distinct forces for the two different binding modes, with mode A dissociating at ~1020 pN, and mode B at ~595 pN, both at a 5.0 Å/ns pulling speed ( Fig.…”

“…At the computational level, these experimental single-molecule approaches can be elaborated upon by employing molecular dynamics (MD) simulations 34 . When combined, these experimental and computational approaches can provide mechanistic insights into the dynamics of receptor-ligand complexes 26,35 .Here we investigated a complex called XMod-Doc:Coh responsible for anchoring the Rc cellulosome complex to the bacterial cell wall and used AFM-SMFS, smFRET, and MD simulations to study putative dual-binding modes and catch bond behavior of the complex. We developed a state map with experimentally transition rates to fully describe the system, and performed kinetic Monte Carlo simulations that recapitulate the experimental data.…”

“…At the computational level, these experimental single-molecule approaches can be elaborated upon by employing molecular dynamics (MD) simulations 34 . When combined, these experimental and computational approaches can provide mechanistic insights into the dynamics of receptor-ligand complexes 26,35 .…”