A defining characteristic of complex life is the capacity to integrate a variety of stimuli from the environment and, through the interaction of a dizzying array of biomolecules, to properly interpret and respond to such stimuli. Gene duplications and random mutations, when filtered through the sieve of natural selection, provides living organisms with the creative power needed to produce the suite of highly specialized molecules required to perform the vital task of sensing environmental cues. From maintaining balance by harnessing the power of gravity in vertebrates, to the detection of a dangerous shift from diffusive equilibrium in yeast cells, proteins play a vital role in detecting changes in the environment of all living organisms.
Central to the function of many of these proteins is the transmission or detection of mechanical forces applied to the proteins themselves, or to surrounding cellular structures and tissues in which they are embedded. Such force sensitive proteins are thus ideal subjects of single-molecule, quantitative biophysical approaches to better understand the molecular basis of force transduction. Examples of such proteins are cadherin-23 (CDH23) and protocadherin-15 (PCDH15), two proteins required for vertebrate hearing; desmoglein (DSG) and desmocollin (DSC), two proteins that are vital for maintaining tissue integrity in the presence of constant mechanical stress; and transient receptor potential yeast 1 (TRPY1), a mechanically-sensitive ion channel in yeast that restores osmotic balance in response to hyperosmotic shock. While these proteins are involved in unrelated biological processes, they all have evolved the ability to detect and respond to force generated by environmental sources, and their proper function is vital to the survival of the host organism. Here, a multidisciplinary approach is used to provide fundamental insights into the structure, mechanical properties, and dynamics of these specific proteins, as well as into protein-protein interactions and the biophysics of force transduction in general.
CDH23 and PCDH15 interact in the inner ear of all vertebrates to form the tip link, a fine protein filament essential for transducing the force generated by sound waves. Their interaction must be strong enough to withstand the constant and repeated application of force, but must also be able to break and re-form in response to potentially damaging stimuli. Additionally, this complex is likely under varying selection pressures depending on the lifestyle and environment of each organism, and thus presents an ideal system to study the evolutionary biophysics of force-transducing protein-protein interactions. High-resolution structures obtained through X-ray crystallography reveal a high degree of structural similarity in these proteins from evolutionarily distant vertebrates, indicating that their architecture has been maintained throughout evolution despite significant sequence divergence. Through the use of all-atom and coarse-grained molecular dynamics (MD) simulations, it was discovered that these evolutionary sequence changes result in significant differences in the dynamics, forces, and energies of unbinding involved in this complex, and that these differences are supported by affinities obtained through surface plasmon resonance experiments. It is hypothesized here that such differences reflect a varying degree of purifying selection in the different vertebrate lineages, and this is supported by biophysics-based molecular sequence evolution simulations as well as evolutionary rate analyses. These results are likely not specific to CDH23 and PCDH15, and may shape the biophysics-based evolutionary landscape of other protein-protein complexes as well.
As with CDH23 and PCDH15, DSG and DSC are members of the cadherin superfamily of proteins and interact with one another to form a cell-cell contact that must withstand force. That is where the similarity ends between these systems, however, as DSC and DSG not only exhibit different overall structures from CDH23 and PCDH15, but they coalesce in a lattice of DSG and DSC molecules to form a robust junction between cells called the desmosome. Additionally, their mode of interaction is entirely distinct from CDH23 and PCDH15. Found in cardiac and epithelial tissue, these proteins are vital for maintaining a robust connection between cells despite abrasions and stretching forces that these tissues are subjected to. Through MD simulations, the mechanical properties of these proteins has been described both in dimers, as well as in a hypothetical model of the desmosomal lattice. Through comparison of these two systems, the simulations reveal the role that parallel cis-interactions play in the forces and mechanics of the lattice, and provide a model for how the desmosome may function in vivo. Results of these simulations demonstrate how desmosomal proteins exhibit a similar mechanical response to E-cadherins but a distinct response from the clustered protocadherins, provide insight into the role of cell-cell junctions in tissue morphogenesis and wound healing as well as into the molecular basis of disease mutations, and can be used as a guide for future experimental design.
While the ion channel TRPY1 is not directly involved in force transduction as with the CDH23-PCDH15 and DSG-DSC systems, it has evolved to respond to forces generated through the stretching or compression of the membrane in which it is embedded. TRPY1 has been characterized experimentally, and its activity has been found to be modulated by the binding of both cytoplasmic and luminal calcium (Ca2+), binding of lipid molecules, and membrane stretch. However, the molecular mechanisms involved in these processes are not well understood. The first structure of TRPY1, obtained by collaborators, facilitated MD simulations that revealed varying degrees of stability and dynamics in essential structural domains depending on whether Ca2+ and/or the regulatory lipids were bound to the protein. Analysis of these simulations also reveals the paths of allosteric communication between the transmembrane and cytoplasmic regions, and how removal of inhibitory ligands affect them. To ensure these results are not an effect of insufficient sampling, µs long simulations were ran, and the results of these longer simulations corroborate the conclusions of the shorter simulations. Finally, to obtain a model of the open state, the pore was forced open by an expanding cylinder, applying forces to the protein radially from the center of the pore, after which an external electric field was applied to simulate a membrane voltage. Permeation of K+ across the pore was observed from which a conductivity of 480 pS was calculated, which is similar to experimentally obtained values. These results provide an insight into the function and modulation of the TRPY1 protein as well as molecular insight into the activation of mechanically-gated ion channels in general.