The dual-function disabled-1 PTB domain exhibits site independence in binding phosphoinositide and peptide ligands.
While typical intracellular protein modules have only one ligand-binding site, there are rare examples of single modules that bind two different ligands at distinct binding sites. Here we present a detailed mutational and energetic analysis of one such domain, the phosphotyrosine binding (PTB) domain of Disabled-1 (Dab1), which binds to both peptide and phosphoinositide (PI) ligands simultaneously at structurally distinct binding sites. Through the techniques of isothermal titration calorimetry (ITC), analysis of Dab1 PTB domain mutants, and nuclear magnetic resonance (NMR), we have evaluated the characteristics of binding of the Dab1 PTB domain to various peptide and PI ligands. These studies reveal that the presence of saturating concentrations of one ligand has little effect on the binding constant for a second ligand at the other site. In addition, proteins with single-point mutations in the peptide-binding site retain native affinity for PI ligands, while proteins with mutations that prevent PI binding retain native affinity for peptide. NMR titrations show that the final structure of the ternary complex is the same independent of the order of addition of the two ligands. Together, these studies show that binding of peptide and PI ligands is energetically independent and noncooperative.
Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Peggy C
PC
Stolt
Didem
D
Vardar
Stephen C
SC
Blacklow
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D002942:Circular Dichroism; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013816:Thermodynamics
C103773:Dab1 protein, mouse; D008024:Ligands; D009419:Nerve Tissue Proteins; D010455:Peptides; D010716:Phosphatidylinositols; D019000:Phosphotyrosine