Synaptotagmin-like protein (Slp) homology domain 1 of Slac2-a/melanophilin is a critical determinant of GTP-dependent specific binding to Rab27A.
The N-terminal synaptotagmin-like protein (Slp) homology domain (SHD) of the Slp and Slac2 families has recently been identified as a specific Rab27A-binding domain (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218; Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432-12436). The SHD consists of two conserved alpha-helical regions (SHD1 and SHD2) that are often separated by two zinc finger motifs. However, the structural basis of Rab27A recognition by the SHD (i.e. involvement of each region (SHD1, zinc finger motifs, and SHD2) in Rab27A recognition and critical residue(s) for Rab27A/SHD interaction) had never been elucidated. In this study, systematic deletion analysis and Ala-based site-directed mutagenesis showed that SHD1 of Slac2-a/melanophilin alone is both necessary and sufficient for high affinity specific recognition of the GTP-bound form of Rab27A. By contrast, the zinc finger motifs and SHD2 are not an autonomous Rab27A-binding site and seem to be important for stabilization of the structure of the SHD or higher affinity Rab27A binding. In addition, chimeric analysis of Rab3A and Rab27A showed that the specific sequence of the switch II region of Rab27 isoforms (especially Leu-84, Phe-88, and Asp-91 of Rab27A), which is not conserved in the Rab3 or Rab8 isoforms, is essential for recognition by the Slac2-a SHD. Based on these findings, I propose that SHD1 of the Slp and Slac2 families be referred to as RBD27 (Rab-binding domain specific for Rab27 isoforms).
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN (Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry*; D017353:Gene Deletion; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D033921:Vesicular Transport Proteins; D016335:Zinc Fingers; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C492410:MLPH protein, human; D008565:Membrane Proteins; C435724:Mlph protein, mouse; D020033:Protein Isoforms; D011994:Recombinant Proteins; C424133:Sytl1 protein, mouse; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C066694:RAB27A protein, human; C082377:RAB8A protein, human; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins