Journal article
Structural and thermodynamic analysis of PDZ-ligand interactions
Methods in enzymology, Vol.488(C), pp.81-100
2011
DOI: 10.1016/B978-0-12-381268-1.00004-5
PMCID: PMC4137555
PMID: 21195225
Abstract
Tiam-family guanine exchange proteins are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These modular proteins contain a variety of signaling domains, including a single postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Here, we show how structural and thermodynamic approaches applied to the Tiam1 PDZ domain can be used to gain unique insights into the affinity and specificity of PDZ-ligand interactions with peptides derived from Syndecan1 and Caspr4 proteins. First, we describe a fluorescence anisotropy-based assay that can be used to determine PDZ-ligand interactions, and describe important considerations in designing binding experiments. Second, we used site-specific mutagenesis in combination with double-mutant cycle analysis to probe the binding energetics and cooperativity of residues in two ligand binding pockets (S(0) and S(-2)) that are involved in Tiam1 PDZ-ligand interactions. Peptide ligand binding results and double-mutant cycle analysis revealed that the S(0) pocket was important for Syndecan1 and Caspr4 peptide interactions and that the S(-2) pocket provided selectivity for the Syndecan1 ligand. Finally, we devised a "peptide evolution" strategy whereby a Model consensus peptide was "evolved" into either the Syndecan1 or Caspr4 peptide by site-directed mutagenesis. These results corroborated the PDZ mutational analysis of the S(0) pocket and identified the P(-4) position in the ligand as critical for Syndecan1 affinity and selectivity. Together, these studies show that a combined structural and thermodynamic approach is powerful for obtaining insights into the origin of Tiam1 PDZ-ligand domain affinity and specificity.
Details
- Title: Subtitle
- Structural and thermodynamic analysis of PDZ-ligand interactions
- Creators
- Tyson R Shepherd - Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USAErnesto J Fuentes
- Resource Type
- Journal article
- Publication Details
- Methods in enzymology, Vol.488(C), pp.81-100
- DOI
- 10.1016/B978-0-12-381268-1.00004-5
- PMID
- 21195225
- PMCID
- PMC4137555
- NLM abbreviation
- Methods Enzymol
- ISSN
- 0076-6879
- eISSN
- 1557-7988
- Publisher
- United States
- Grant note
- GM067795 / NIGMS NIH HHS T32 GM067795 / NIGMS NIH HHS T32 GM008365 / NIGMS NIH HHS
- Language
- English
- Date published
- 2011
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984024403702771
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