Computer-aided design of a PDZ domain to recognize new target sequences

PDZ domains are small globular domains that recognize the last 4–7 amino acids at the C-terminus of target proteins. The specificity of the PDZ–ligand recognition is due to side chain–side chain interactions, as well as the positioning of an α-helix involved in ligand binding. We have used computer-aided protein design to produce mutant versions of a Class I PDZ domain that bind to novel Class I and Class II target sequences both in vitro and in vivo, thus providing an alternative to primary antibodies in western blotting, affinity chromatography and pull-down experiments. Our results suggest that by combining different backbone templates with computer-aided protein design, PDZ domains could be engineered to specifically recognize a large number of proteins.

[1]  S. Fields,et al.  The two-hybrid system: an assay for protein-protein interactions. , 1994, Trends in genetics : TIG.

[2]  John H. Lewis,et al.  Crystal Structures of a Complexed and Peptide-Free Membrane Protein–Binding Domain: Molecular Basis of Peptide Recognition by PDZ , 1996, Cell.

[3]  T. Südhof,et al.  CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[4]  Ron D. Appel,et al.  Protein identification with sequence tags , 1996, Current Biology.

[5]  P. S. Kim,et al.  Context-dependent secondary structure formation of a designed protein sequence , 1996, Nature.

[6]  T. Südhof,et al.  Binding of neuroligins to PSD-95. , 1997, Science.

[7]  L. Cantley,et al.  Recognition of Unique Carboxyl-Terminal Motifs by Distinct PDZ Domains , 1997, Science.

[8]  Ann Marie Craig,et al.  CRIPT, a Novel Postsynaptic Protein that Binds to the Third PDZ Domain of PSD-95/SAP90 , 1998, Neuron.

[9]  G. Lyons,et al.  Expression of the Mitotic Motor Protein Eg5 in Postmitotic Neurons: Implications for Neuronal Development , 1998, The Journal of Neuroscience.

[10]  C. Hovens,et al.  Mutagenesis and selection of PDZ domains that bind new protein targets , 1999, Nature Biotechnology.

[11]  Mingjie Zhang,et al.  Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide , 1999, Nature Structural Biology.

[12]  Shoshana Wodak,et al.  Protein design: a computer-based approach , 1999 .

[13]  V. Tumanyan,et al.  Amino acid composition of protein termini are biased in different manners. , 1999, Protein engineering.

[14]  Luis Serrano,et al.  A tale of two secondary structure elements: when a β-hairpin becomes an α-helix 1 1Edited by A. R. Fersht , 1999 .

[15]  L Serrano,et al.  A tale of two secondary structure elements: when a beta-hairpin becomes an alpha-helix. , 1999, Journal of molecular biology.

[16]  M. Teresa Pisabarro,et al.  Analysis of PDZ Domain-Ligand Interactions Using Carboxyl-terminal Phage Display* , 2000, The Journal of Biological Chemistry.

[17]  G. Kozlov,et al.  Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor. , 2000, Biochemistry.

[18]  L Serrano,et al.  Computer-aided design of beta-sheet peptides. , 2001, Journal of molecular biology.

[19]  Luis Serrano,et al.  Computational estimation of specific side chain interaction energies in α helices , 2001 .

[20]  W. Lim,et al.  Mechanism and role of PDZ domains in signaling complex assembly. , 2001, Journal of cell science.

[21]  M. Sheng,et al.  PDZ domains and the organization of supramolecular complexes. , 2001, Annual review of neuroscience.

[22]  Technical tips online , 2002 .