Functional reassembly of a split PH domain.
The pleckstrin homology (PH) domain forms a structurally conserved protein module of approximately 120 amino acid residues. Several proteins involved in cellular signaling and cytoskeletal organization possess split PH domains while their biological roles and ligand binding activity remain to be clarified. We have designed a split PH domain from a structurally well-characterized PH domain of phospholipase Cdelta(1) by dissecting the PH domain and tethering a coiled coil module to each subunit to ask a question of whether the coiled coil could mediate a functional reassembly of the split PH domain. Isothermal titration microcalorimetry measurements indicated a formation of a thermodynamically stable 1:1 complex of the N-terminal and C-terminal halves of the split PH domain by the coiled coil formation. The reassembled split PH domain binds to IP(3), a target molecule of the parent PLCdelta(1) PH domain, but not to L-IP(3), indicating that the split PH domain maintains a binding selectivity similar to the native PLCdelta(1) PH domain. These results demonstrate that the split PH domain folds into a functional structure when the split halves are brought to close proximity, and suggest that the native split PH domains, such as found in PLCgamma(1), have distinctive functions upon the reassembly.
Sugimoto
Kenji
K
Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.
Mori
Yasuo
Y
Makino
Keisuke
K
Ohkubo
Katsutoshi
K
Morii
Takashi
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D054803:Phospholipase C delta; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
D007527:Isoenzymes; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D054803:Phospholipase C delta