Technical Highlight - November 2011
Short description: A systems approach reveals an unexpectedly high frequency of PDZ domain dimerization.
Protein-protein complexes mediate most cellular processes, and proteome-wide quantification of these interactions is an important goal for systems biology. The availability of genome sequences has shown that many proteins are composed of relatively few modular domains. One example is the ubiquitous PDZ domain that typically binds to the C-terminal tails of target proteins, although there are a few examples of noncanonical PDZ-PDZ dimerization.
MacBeath and colleagues explored the extent of PDZ dimerization, using a systems approach to quantify interactions within a set of 157 PDZ domains from the mouse genome. Guided by sequence alignments, the authors designed expression constructs and prepared microarrays containing functional PDZ domains and a Cy3 fluorophore.
A pilot experiment probed the interactions of 96 diverse PDZ domains with the PDZ domain from nNOS, labeled with a Cy5 fluorophore. Using a relatively low threshold for false negatives, five specific PDZ-PDZ interactions were identified, three of which had been previously reported. To confirm these interactions, the authors established a fluorescence polarization (FP) assay that allowed them to measure the Kd for each binding event. They confirmed all five interactions by the FP assay, and the Kd values they obtained agreed with known values in the two cases for which data were available.
Next, the authors examined the 157 mouse PDZ domains and identified 314 unique PDZ-PDZ interactions, with 46 of the 157 PDZ domains (∼30%) participating in at least one interaction. Positive hits with Kd <25 μM were selected for further analysis: 37 PDZ-PDZ interactions were confirmed, including 33 novel ones. As protein domains can function differently in the context of the full-length protein, they tested a set of 11 PDZ-PDZ interactions with Kd <5 μM for association in vivo. Using co-affinity purification, all 11 interactions were detected, including seven novel ones. Finally, database searches supported the functional significance of these interactions, and the authors suggest that PDZ dimerization can contribute to the specificity of protein complexes. Overall, the study demonstrates the potential of exploring the proteome using known interaction modules.
B.H. Chang et al. A systematic family-wide investigation reveals that ∼30% of mammalian PDZ domains engage in PDZ-PDZ interactions.
Chem. Biol. 18, 1143-1152 (2011). doi:10.1016/j.chembiol.2011.06.013