Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a

Scientific Reports, Jun 2015

The tail-inhibition model is generally accepted for the regulation of myosin-5a motor function. Inhibited myosin-5a is in a folded conformation in which its globular tail domain (GTD) interacts with its head and inhibits its motor function, and high Ca2+ or cargo binding may reduce the interaction between the GTD and the head of myosin-5a, thus activating motor activity. Although it is well established that myosin-5a motor function is regulated by Ca2+, little is known about the effects of cargo binding. We previously reported that melanophilin (Mlph), a myosin-5a cargo-binding protein, is capable of activating myosin-5a motor function. Here, we report that Mlph-GTBDP, a 26 amino-acid-long peptide of Mlph, is sufficient for activating myosin-5a motor function. We demonstrate that Mlph-GTBDP abolishes the interaction between the head and GTD of myosin-5a, thereby inducing a folded-to-extended conformation transition for myosin-5a and activating its motor function. Mutagenesis of the GTD shows that the GTD uses two distinct, non-overlapping regions to interact with Mlph-GTBDP and the head of myosin-5a. We propose that the GTD is an allosteric protein and that Mlph allosterically inhibits the interaction between the GTD and head of myosin-5a, thereby activating myosin-5a motor function.

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Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a

Abstract The tail-inhibition model is generally accepted for the regulation of myosin-5a motor function. Inhibited myosin-5a is in a folded conformation in which its globular tail domain (GTD) interacts with its head and inhibits its motor function, and high Ca2+ or cargo binding may reduce the interaction between the GTD and the head of myosin-5a, thus activating motor activity. Although it is well established that myosin-5a motor function is regulated by Ca2+, little is known about the effects of cargo binding. We previously reported that melanophilin (Mlph), a myosin-5a cargo-binding protein, is capable of activating myosin-5a motor function. Here, we report that Mlph-GTBDP, a 26 amino-acid-long peptide of Mlph, is sufficient for activating myosin-5a motor function. We demonstrate that Mlph-GTBDP abolishes the interaction between the head and GTD of myosin-5a, thereby inducing a folded-to-extended conformation transition for myosin-5a and activating its motor function. Mutagenesis of the GTD shows that the GTD uses two distinct, non-overlapping regions to interact with Mlph-GTBDP and the head of myosin-5a. We propose that the GTD is an allosteric protein and that Mlph allosterically inhibits the interaction between the GTD and head of myosin-5a, thereby activating myosin-5a motor function. Introduction Class V myosin (Myo5) is one of the oldest classes of myosins, which is distributed from lower eukaryotes, such as yeast, to vertebrate cells1. Thus far, the most well-characterized Myo5 is vertebrate Myo5a, which is a processive motor that is capable of individually moving along an actin filament for several steps without dissociation2,3,4,5,6. Myo5a contains a motor domain and an extended lever arm followed by a coiled-coil dimerizing region and a C-terminal globular tail domain (GTD)7. Myo5a is responsible for the transportation and localization of a number of vesicles, including melanosomes in melanocytes (for a review, see8). Melanosomes associate with Myo5a via Rab27a and melanophilin (Mlph)9,10,11. Rab27a localizes to melanosome membranes and interacts with Mlph. Mlph contains an N-terminal Rab27a-binding domain and two independent Myo5a-binding regions9,10. The first interaction occurs between the melanocyte-specific exon-F in the Myo5a tail and Mlph-EFBD (Exon-F Binding Domain; residues 241–400), and the second interaction occurs between the GTD of Myo5a and Mlph-GTBD (Globular Tail domain-Binding Domain; residues 147–240)12. Spudich and colleagues narrowed down Mlph-GTBD to a 26-residue peptide (residues 176–201)13. Cell biology studies have demonstrated that both the interaction between exon-F and Mlph-EFBD and the interaction between the GTD and Mlph-GTBD are essential for rescuing the melanosome transport defect in dilute and leaden melanocytes9,14. A critical question is how the motor function of Myo5a is regulated. A tail-inhibition model for Myo5a regulation is generally accepted. In this model, Myo5a in the inhibited state is in a folded conformation such that its tail interacts with its head and inhibits motor activity, and high Ca2+ or cargo binding may reduce the interaction between the head and tail, thus activating motor activity15,16,17. Ca2+ activation of Myo5a’s motor function has been the subject of intense investigation15,16,17,18,19,20,21,22,23. Ca2+-induced activation of Myo5a’s ATPase activity is accompanied by a folded-to-extended conformation transition16. Truncation analyses of Myo5a have indicated that Myo5a motor function is inhibited by the GTD, and this inhibition is abolished by Ca2+19,20. We recently found that the calmodulin (CaM) in the first IQ motif participates in the interaction between the head and the GTD and is responsible for the activation of Myo5a by Ca2+24. Thus, it is likely that Ca2+ induces a conformational change in the CaM in IQ1, thereby preventing an interaction between the head and the GTD and causing motor function activation. However, little is known about the effects of cargo binding on the motor function of Myo5a. Because the tail of Myo5a not only functions as a cargo binding site but also serves as a key regulatory component of Myo5a, Sellers and colleagues proposed that the binding of cargo to the tail might activate the motor activity of Myo5a15. Consistent with this prediction, we found that Mlph directly stimulates the actin-activated ATPase activity of Myo5a25. Recently, Trybus and colleagues demonstrated at the single-molecule level that Mlph significantly increases the number of processively moving Myo5a molecules26. However, it is not clear whether Mlph activates the Myo5a motor by the same mechanism as Ca2+; i.e., by abolishing the tail inhibition of the head. In this study, we found that Mlph-GTBDP, the 26-residue Myo5a-GTD binding peptide of Mlph identified by Spudich and colleagues13, is capable of activating the motor function of Myo5a. We demonstrate that Mlph-GTBDP abolishes the interaction between the GTD and the h (...truncated)


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Lin-Lin Yao, Qing-Juan Cao, Hai-Man Zhang, Jie Zhang, Yang Cao, Xiang-dong Li. Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a, Scientific Reports, 2015, Issue: 5, DOI: 10.1038/srep10874