Energetic determinants of internal motif recognition by PDZ domains.
PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.
Harris
B Z
BZ
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0450, USA.
Hillier
B J
BJ
Lim
W A
WA
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D002135:Calcium-Binding Proteins; D002942:Circular Dichroism; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012996:Solutions; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D008024:Ligands; D008565:Membrane Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D012996:Solutions; C033652:postsynaptic density proteins; C091825:syntrophin alpha1; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I