Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

Abstract

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament (“F-actin”) assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, “ jordan ”). Here, we present cryo–electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate–bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide state–dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

Bending forces and nucleotide state jointly regulate F-actin structure

“…2b,c ). This region, which is well established to be flexible in F-actin 27 , 32 , 40 , 41 , also corresponded to the lowest-resolution region of our maps, and differences may therefore be attributable to resolution-dependent uncertainty in the atomic models. At the helical-lattice level, the refined rise was slightly larger in ADP–F-actin (28.1 Å versus 27.8 Å in ADP-P i –F-actin), which accumulates into detectable differences in subunit positioning at longer length scales (Extended Data Fig.…”

“…The ADP–F-actin sample corresponds to a pre-existing dataset described in a recent study 41 . ADP–F-actin was prepared as described above, except KH 2 PO 4 was omitted and KMEI buffer (50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 10 mM imidazole pH 7.0, 1 mM DTT) + 0.01% NP40 substitute was used instead of KMEH.…”

“…For the ADP-P i –F-actin dataset, beam-image shift was used to collect 4,834 single exposures from nine holes in a 3-by-3 grid per each stage translation. For the ADP–F-actin dataset, which has previously been reported 41 and was reprocessed here, 1,548 exposures were directly targeted using stage translations with a single exposure per hole.…”

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin

“…The ADP-coordination described above closely matches the active site in the crystal structure of the actin-free “post-rigor” bovine β-cardiac myosin in complex Mg 2+ · ADP (Robert-Paganin et al, 2018). Comparing these active site architectures with published coordinates of other Mg 2+ · ADP complexed actomyosin isoforms, such as myosin V, 1b, and 15, we find that they are nearly superimposable, highlighting the evolutionary conservation of this part of the myosin structure (Mentes et al, 2018; Pospich et al, 2021; Gong et al, 2022).…”

“…Cryo-EM methods have been used to generate reconstructions of many actomyosin complexes, including ones for mammalian bovine and porcine cardiac myosin(Doran et al, 2020; Risi et al, 2021). However, only three high-resolution cryo-EM solutions have been solved for ADPassociated actomyosin, all containing non-sarcomeric myosins (containing myosin V, myosin IB, and myosin 15) and hence none including striated muscle myosin isoforms(Pospich et al, 2021; Gong et al, 2022; Mentes et al, 2018). In the current study, we focused on correcting this deficit by reconstructing high-resolution structures of F-actin decorated with mammalian cell expressed β-cardiac myosin II under nucleotide-free conditions or in the presence of Mg 2+ · ADP.…”