Highlights d TMC1/2 cannot form functional mechanotransduction channels in hair cells without TMIE d TMIE is a subunit of the mechanotransduction channel of cochlear hair cells d TMIE mutations affect pore properties such as conductance and ion selectivity d TMIE binds PIP 2 , and deafness mutations affect PIP 2 binding and transduction
Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.
Summary
The tip link, a filament formed by protocadherin 15 (Pcdh15) and cadherin 23, conveys mechanical force from sound waves and head movement to open hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here we biophysically and structurally characterize the unusual cis-homodimeric architecture of Pcdh15. We show that Pcdh15 molecules form double helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric Pcdh15 assembly and reveal the Pcdh15 extracellular domain as a parallel double-helix with cis cross-bridges at the two locations we defined. The helical configuration suggests the potential for elasticity through helix winding/unwinding. Functional studies in hair cells show that mutations that perturb Pcdh15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of Pcdh15, and show that dimerization is critical for sensing mechanical stimuli.
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