Structural analysis of mammalian myosin Va and yeast Myo2 with their cargo adaptors reveals a common mechanism of interaction. (A and C) Electrostatic surface potentials for the yeast and mammalian myosin Va tails, respectively. (B and D) Adaptor peptides each interact with the longest helix of the myosin V tail. (E) Ribbon representation illustrates that adaptor peptides adopt similar folds and structurally overlap when aligned to the longest myosin V helix (not shown). Sequence alignment reveals common features within the adaptors that are important for myosin V recognition. Orange indicates hydrophobic residues, and blue represents positively charged residues. Underlined residues are critical for interaction (Pylypenko et al., 2013, 2016; Wei et al., 2013; Tang et al., 2019; this study). Note that Vac17 is the only adaptor that interacts with myosin V in an antiparallel orientation. (F) Vac17(L137) and Mmr1(410) each form hydrophobic interactions with Myo2(L1229). Hydrophobic contacts are further stabilized by ionic interactions between Vac17(142)–Myo2(E1222) and Mmr1(R409)–Myo2(E1293). Asterisk indicates the suppressor residue Vac17(I140), which is spatially proximal to Myo2(L1229). Cyan dashes indicate bond distances between 2.5 and 5 Å. (G) Mlph(F196) interacts with myosin Va(I1535), a key residue mediating hydrophobic contacts (Wei et al., 2013; Pylypenko et al., 2013). Similarly, Spir2(L414) is predicted to form hydrophobic interactions with myosin Va(I1535). In addition, hydrogen bonds are formed between myosin Va(R1528) and Mlph(S190), between myosin Va(E1595) and Mlph(F191), and between myosin Va(E1595)-Spir2(A411). These polar interactions are analogous to the charged contacts between Myo2(E1222) and Vac17(R142) and between Myo2(E1293) and Mmr1(R409).