Abstract
PDZ domain proteins control multiple cellular functions by governing assembly of protein complexes. It remains unknown why individual PDZ domains can bind the extreme C terminus of very diverse binding partners and maintain selectivity. By employing NMR spectroscopy, together with molecular modeling, mutational analysis, and fluorescent polarization binding experiments, we identify here three structural mechanisms explaining why the PDZ domain of PICK1 selectively binds >30 receptors, transporters, and kinases. Class II ligands, including the dopamine transporter, adopt a canonical binding mode with promiscuity obtained via differential packing in the binding groove. Class I ligands, such as protein kinase Cα, depend on residues upstream from the canonical binding sequence that are likely to interact with flexible loop residues of the PDZ domain. Finally, we obtain evidence that the unconventional ligand ASIC1a has a dual binding mode involving a canonical insertion and a noncanonical internal insertion with the two C-terminal residues forming interactions outside the groove. Together with an evolutionary analysis, the data show how unconventional binding modes might evolve for a protein recognition domain to expand the repertoire of functionally important interactions.
Highlights
The molecular basis for how the PDZ domain of the scaffolding protein protein interacting with C kinase 1 (PICK1) selectively binds Ͼ30 different ligands is unclear
Expression, and Purification of PICK1 PDZ Constructs—A PICK1 PDZ-3C-GluA2 construct consisting of the PICK1 residues 18 –110, a linker region with a protease 3C cleavage site (LEVLFGGP), and the residues (VYGIESVKI) derived from the C terminus of the AMPA receptor subunit 2 was purchased from Origene, Germany, and subcloned into the pGEX4T2 vector (Novagen) producing an N-terminal glutathione S-transferase (GST) fusion construct [34]
PICK1 PDZ Forms Stable Structures Only with Class II Ligands—To understand the binding modes in atomic detail, we analyzed by NMR spectroscopy seven constructs of PICK1 PDZ with C-terminal extensions corresponding to the 10 C-terminal residues of the ligands PKC␣, GluA2, human epidermal growth factor receptor 2 (HER2), ASIC1a, UNC5H1, dopamine transporter (DAT), and GLT1b
Summary
The molecular basis for how the PDZ domain of the scaffolding protein PICK1 selectively binds Ͼ30 different ligands is unclear. PDZ domains are ϳ90 residues long and folded to form an elongated groove consisting of mainly two pockets (S0 and SϪ2) that, with very few exceptions, bind the last 3– 4 C-terminal residues of the target proteins With such a short binding sequence, it has been a central question how sufficient specificity of PDZ interactions is ensured in a cellular environment [3,4,5,6,7,8]. A large study of mouse PDZ domains suggested that PDZ domains do not fall into discrete classes but instead lie on a continuum with the ligand selectivity derived from interactions throughout the binding pocket [5] Another comprehensive study supported that PDZ domains form at least 16 different specificity classes recognizing up to seven C-terminal ligand residues [7].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
