Vertebrates rely on the inner ear to convert the mechanical stimuli of sound and head movements into electrical signals that can be decoded by the brain. This process, called “mechanotransduction”, relies on a finely tuned molecular machine located in inner ear hair cells. Central to this machine is the hair-cell tip link, a protein filament that pulls open mechanosensitive ion channels, allowing for the depolarization of hair cells and the subsequent generation of an electrical signal. In a paper recently published in Structure (PDF), a team from Rachelle Gaudet’s lab – former Harvard Chemical and Physical Biology undergraduate Robert Powers ’14 and former postdoctoral fellow Marcos Sotomayor (now at Ohio State University) – has determined crystal structures of a protein that makes up the essential tip link. The structures, along with accompanying molecular dynamics simulations, reveal unusual protein characteristics. These results deepen our understanding of mechanotransduction and have broad implications for the study of cadherins, a protein family responsible for cell adhesion and signaling in a variety of biological contexts.
The term “cadherin” signifies calcium-dependent adhesion proteins. Cadherins are generally formed of a repeated array of β-sheet protein structure alternating with calcium-binding linker regions. The classical cadherins then form homodimers that yield strong adhesions between neighboring cells. The tip link comprises the non-classical cadherin proteins protocadherin-15 and cadherin-23. Previous work in the Gaudet lab showed that these two proteins interact via a novel extended “handshake” bond, which allows them to withstand and transmit mechanical forces. Protocadherin-15 and cadherin-23 display unique characteristics that distinguish themselves from other cadherins. In addition to interacting using an unusual handshake bond, sequence analyses revealed that certain regions of each protein lack expected calcium-binding residues. Given that tip link mechanical strength and function is known to depend on calcium, the “missing” calcium-binding residues raised several questions: Can the protein regions that lack the conserved residues still bind calcium? How does the potential lack of calcium impact the dynamics and mechanical strength of the proteins?
To answer this question, Robert Powers, now a graduate student at Harvard Medical School in the Biophysics Program, determined the crystal structure of a fragment of protocadherin-15 that lacks several of the calcium-binding residues. The structures revealed that instead of binding three calcium ions like classical cadherins, this protocadherin-15 fragment contains a linker region that binds only two calcium ions using a novel coordination mechanism. The crystal structure also suggested that the presence of only two calcium ions results in increased protein flexibility.
To confirm this, the team performed molecular dynamics simulations at the Ohio Supercomputer Center to observe and quantify the flexibility of the protocadherin-15 fragment. The molecular motion trajectories showed that the linker that binds only two calcium ions is indeed more flexible than a linker that binds the canonical three calcium ions, but still more rigid than a calcium-free linker. The simulations also revealed that this increased flexibility does not come at the cost of mechanical strength. These results demonstrate that the calcium-binding stoichiometry of cadherins can modulate their dynamics. In the context of the tip link, increased flexibility in particular regions may be important for the efficient transduction of mechanical forces. As a simple analogy, the new structure and sequence analyses suggest that the tip link has several flexing elbows, and may thus behave more like a flexible rope than a stiff rod.
Adding to the intrigue of this particular protocadherin-15 fragment was previous work by Pardis Sabeti’s (OEB) lab that showed that within this unique linker region there is a single amino acid substitution that is under positive selection in East Asian populations, suggesting that this variation somehow confers a competitive advantage. Robert Powers determined the structure of both the ancestral and derived variants and observed no significant changes in the structure or the dynamics of the protein. Thus, while the nature of the evolutionary advantages remains unknown, the findings suggest that the advantages may stem from transcriptional changes or protein folding changes rather than from the biochemical and biophysical properties of the protein.
In summary, the new structures and simulations of protocadherin-15 provide valuable insights into the non-canonical characteristics of some cadherin proteins and how these unique features may allow certain molecules to perform their distinctive roles in processes that make multicellular life possible.
by Robert Powers, Marcos Sotomayor, and Rachelle Gaudet
