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J. Biol. Chem., Vol. 282, Issue 29, 21518-21528, July 20, 2007

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Identification of a Minimal Myosin Va Binding Site within an Intrinsically Unstructured Domain of Melanophilin^*

From the Department of Biochemistry, Stanford University, Stanford, California 94041

Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and ¹H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.

Received for publication, March 6, 2006 , and in revised form, May 10, 2007.

^* This work was supported in part by National Institutes of Health Grant PO1 AR42895 (to J. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Howard Hughes Medical Institute predoctoral fellowship.

² To whom correspondence should be addressed. E-mail: jspudich{at}stanford.edu.

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