Structural and membrane binding properties of the prickle PET domain.
The planar cell polarity (PCP) pathway is required for fetal tissue morphogenesis as well as for maintenance of adult tissues in animals as diverse as fruit flies and mice. One of the key members of this pathway is Prickle (Pk), a protein that regulates cell movement through its association with the Dishevelled (Dsh) protein. Pk presents three LIM domains and a PET domain of unknown structure and function. Both the PET and LIM domains control membrane targeting of Dsh, which is necessary for Dsh function in the PCP pathway. Here, we show that the PET domain is monomeric and presents a nonglobular conformation with some properties of intrinsically disordered proteins. The PET domain adopts a helical conformation in the presence of 2,2,2-trifluoroethanol (TFE), a solvent known to stabilize hydrogen bonds within the polypeptide backbone, as analyzed by circular dichroism (CD) and NMR spectroscopy. Furthermore, we found that the conserved and single tryptophan residue in PET, Trp 536, moves to a more hydrophobic environment when accompanied with membrane penetration and that the protein becomes more helical in the presence of lipid micelles. The presence of LIM domains, downstream of PET, increases protein folding, thermostability, and tolerance to limited proteolysis. In addition, pull-down and tryptophan fluorescence analyses suggest that the LIM domains physically interact to regulate membrane penetration of the PET domain. The findings reported here favor a model where the PET domain is engaged in Pk membrane insertion, whereas the LIM domains modulate this function.
Sweede
Matthew
M
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Ankem
Gayatri
G
Chutvirasakul
Boonta
B
Azurmendi
Hugo F
HF
Chbeir
Souhad
S
Watkins
Justin
J
Helm
Richard F
RF
Finkielstein
Carla V
CV
Capelluto
Daniel G S
DG
D000465:Algorithms; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D060588:LIM Domain Proteins; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D014270:Trifluoroethanol / Q000737:chemistry
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D060588:LIM Domain Proteins; D010455:Peptides; D012997:Solvents; C121600:pk protein, Drosophila; D014270:Trifluoroethanol