Molecular analysis of the interaction between cardosin A and phospholipase D(alpha). Identification of RGD/KGE sequences as binding motifs for C2 domains.
Here we report the identification of phospholipase Dalpha as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase Dalpha constructs were performed. Results revealed that the C2 domain of phospholipase Dalpha contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dalpha can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains.
Simões
Isaura
I
Departamento de Biologia Molecular e Biotecnologia, Centro de Neurociências e Biologia Celular, Universidade de Coimbra and Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal.
Mueller
Eva-Christina
EC
Otto
Albrecht
A
Bur
Daniel
D
Cheung
Alice Y
AY
Faro
Carlos
C
Pires
Euclides
E
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D031942:Cynara / Q000201:enzymology*; D005982:Glutathione Transferase / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011058:Pollen / Q000201:enzymology*; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D009842:Oligopeptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C047981:arginyl-glycyl-aspartic acid; D005982:Glutathione Transferase; D010739:Phospholipase D; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus