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

Abstract

SummaryThe motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and myosin-15 mutations cause profound deafness. In a companion study, we report that myosin-15 nucleates actin filament (“F-actin”) assembly and identify a progressive hearing loss mutation (p.D1647G, “jordan”) which disrupts stereocilia elongation by inhibiting actin polymerization. Here, we present cryo-EM structures of myosin-15 bound to F-actin, providing a framework for interpreting deafness mutations and their impacts on myosin-stimulated actin assembly. 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. ADP-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.

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

“…The coordination of Mg 2+ -ADP in our structure closely resembles the ones reported for the strong-ADP state of myosin-IB ( Mentes et al, 2018 ), myosin-VI ( Gurel et al, 2017 ), and myosin-XV ( Gong et al, 2021 ; Figure 1—figure supplement 4 ). Only the position of switch I differs appreciably between myosins, ultimately resulting in varying positions of the coordinated Mg 2+ ion.…”

“… Comparison of the active sites of myosin-V in the strong-ADP state (orange) with the ones of ( A ) myosin-IB (PDB: 6C1D; Mentes et al, 2018 ), ( B ) myosin-VI (PDB: 6BNQ, nucleotide not modeled; Gurel et al, 2017 ), and ( C ) myosin-XV (PDB: 7R91, Gong et al, 2021 ) in the same state (shades of blue, shown as transparent). The coordination of Mg 2+ -ADP is almost identical in all four atomic models.…”

“… Comparison of atomic models of the rigor and strong-ADP states of different actomyosin complexes solved by cryo-EM. ( A ) Superposition of the rigor states of myosin-V (red), myosin-II (PDB: 5H53; Fujii and Namba, 2017 ), myosin-NMIIC (PDB: 5JLH; von der Ecken et al, 2016 ), myosin-NMIIC (PDB: 5JLH; von der Ecken et al, 2016 ), myosin-IB (PDB: 6C1H; Mentes et al, 2018 ), and myosin-XV (PDB: 7R91; Gong et al, 2021 ) (shades of gray), illustrating strongly varying conformations and lever arm orientations. ( B ) Superposition of the strong-ADP states of myosin-V (orange), myosin-IB (PDB: 6C1D; Mentes et al, 2018 ), and myosin-XV (PDB: 7RB8; Gong et al, 2021 ) (shades of gray).…”

“…Instead, cryo electron microscopy (cryo-EM) has proven to be an optimal tool to study filamentous proteins ( Pospich and Raunser, 2018 ) such as the actomyosin complex ( Behrmann et al, 2012 ; von der Ecken et al, 2016 ). To date, the structure of the actomyosin rigor complex has been determined for a variety of myosins ( Banerjee et al, 2017 ; Behrmann et al, 2012 ; Doran et al, 2020 ; Fujii and Namba, 2017 ; Gong et al, 2021 ; Gurel et al, 2017 ; Mentes et al, 2018 ; Risi et al, 2021 ; Robert-Paganin et al, 2021 ; Vahokoski et al, 2020 ; von der Ecken et al, 2016 ). States other than the nucleotide-free rigor state have proven more difficult to study, mainly due to lower binding affinities and short lifetimes.…”

“…States other than the nucleotide-free rigor state have proven more difficult to study, mainly due to lower binding affinities and short lifetimes. In fact, the only other state solved to date is the strong-ADP state; and only two (myosin-IB, myosin-XV) ( Gong et al, 2021 ; Mentes et al, 2018 ) of four independent studies (myosin-Va, myosin-VI) ( Gurel et al, 2017 ; Wulf et al, 2016 ) have achieved high resolution (<4 Å). However, the actin-bound states of myosin, in particular weakly bound transition states for which no structure is yet available, are precisely those that are urgently needed to understand important properties of the myosin motor cycle, such as binding to and detachment from F-actin (recently reviewed in Schröder, 2020 ).…”