Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin.
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 (1)H 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.
Geething
Nathan C
NC
Department of Biochemistry, Stanford University, Stanford, California 94041, USA.
Spudich
James A
JA
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D020460:Melanosomes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry*; D024701:Myosin Type V / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins / Q000737:chemistry; D000075745:rab27 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C435724:Mlph protein, mouse; C513082:Myo5a protein, mouse; D011994:Recombinant Proteins; D000075745:rab27 GTP-Binding Proteins; D024701:Myosin Type V; C512962:Rab27a protein, mouse; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins