Purification and Characterization of Myosin-15, the Molecular Motor Mutated in DFNB3 Human Deafness

Abstract

Stereocilia are mechanosensitive organelles projecting from the surface of inner ear hair cells that detect nanometer deflections induced by sound, gravity or head movement. Unconventional myosin-15 (encoded by Myo15) is hypothesized to regulate stereocilia development by delivering cargoes such as whirlin and Eps8 to their tips. Whether myosin-15 actually functions as a transporter remains untested and little is known regarding its activity, structure and regulation within the highly specialized stereocilia compartment.We have characterized the biochemical kinetics of a subfragment-1 (S1) like truncation of mouse myosin-15, comprising the catalytic ATPase/actin binding domain plus two IQ light chain binding sites. Expression of the recombinant S1 fragment in Sf9 insect cells required co-expression of multiple molecular chaperones in order to recover significant quantities of active protein. Unlike most unconventional myosin classes, the IQ regions of myosin-15 did not bind calmodulin with high affinity; instead preferentially associating with essential (MYL6) and regulatory (MYL12A) light chains that typically partner with myosin-2 isoforms. Single molecule TEM confirmed that the purified S1 was correctly folded and monomeric. The S1 fragment was mechanically active and moved actin filaments at ∼170 nm·s−1 in a gliding motility assay. A powerstroke displacement of 7.8 nm was measured by optical trapping. Transient kinetics revealed that ATP binding to myosin-15 was slow and weak (K1k2 = 0.17 μM−1s−1, 1/K1 = 1898 μM), and that ADP release (∼12s−1) was the rate-limiting step; indicating the predominant steady-state intermediates for myosin-15 would either be with ADP, or no nucleotide bound. Both states bind strongly to actin, suggesting that myosin-15 could be capable of longer-range processive motility if oligomerized in vivo. These data enable future kinetic and structural studies to investigate how deafness-associated mutations targeted within the ATPase ultimately disrupt stereocilia function.