Small-angle X-ray scattering and NMR studies of the conformation of the PDZ region of SAP97 and its interactions with Kir2.1.
The functional localization of potassium inward rectifiers is regulated by SAP97, a PDZ membrane-associated guanylate kinase protein. We describe here an investigation of the conformation of the PDZ domain region of SAP97 PDZ1-3. The NMR and SAXS data reveal conformational dynamics. The NMR data show minimal interdomain contacts, with the U3 linker region between PDZ2 and PDZ3 being largely unstructured. Shape analysis of the SAXS profiles revealed a dumbbell for the PDZ12 double domain. An overall elongated, asymmetric shape comprised of two to three distinct components characterizes the triple domain PDZ1-3. In addition, rigid body modeling shows that the representative average shape does not provide the full picture and that the data for the triple domain are consistent with large variations, suggesting significant conformational flexibility. However, the dynamics appears to be restricted as PDZ3 is located essentially within approximately 40 A from PDZ12. We also show that the Kir2.1 cytoplasmic domain interacts with all three PDZ domains but with a clear preference for PDZ2 even in the presence of the U3 region. We speculate that the restricted dynamics and preferential Kir2.1 binding to PDZ2 are features that enable SAP97 to function as a scaffold protein, allowing other proteins each to bind to the other two PDZ domains in sufficient proximity to yield productive channelosomes.
Goult
Benjamin T
BT
Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom.
Rapley
Jonathan D
JD
Dart
Caroline
C
Kitmitto
Ashraf
A
Grossmann
J Günter
JG
Leyland
Mark L
ML
Lian
Lu-Yun
LY
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000818:Animals; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D054731:PDZ Domains; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D048868:Adaptor Proteins, Signal Transducing; C501119:Dlg1 protein, rat; C489461:Kir2.1 channel; D008565:Membrane Proteins; D024661:Potassium Channels, Inwardly Rectifying