PDZ domains are protein–protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position −2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.

Fas and Fas-associated death domain (FADD) play a critical role in the homeostasis of different cell types. The regulation of Fas and FADD-mediated cell death is pivotal to many physiological functions. The activation of T lymphocytes by concanavalin A (Con A) inhibited Fas-mediated cell death. We identified that among the several activation signals downstream of Con A stimulation, mitogen-activated protein (MAP) kinase kinase (MKK) was the major kinase pathway that antagonized Fas-triggered cell death. MKK1 suppressed FADD- but not caspase-3– induced apoptosis, indicating that antagonism occurred early along the Fas-initiated apoptotic cascade. We further demonstrated that activation of MKK1 led to expression of FLIP, a specific inhibitor of FADD. MKK1 inhibition of FADD-induced cell death was abrogated if induction of FLIP was prevented, indicating that FLIP mediates MKK1 suppression of FADD-mediated apoptosis. Our results illustrate a general mechanism by which activation of MAP kinase attenuates apoptotic signals initiated by death receptors in normal and transformed cells.

Activation of the pDbeta1 promoter at the TCRbeta locus requires a functional distal enhancer, Ebeta. Here, we have analyzed the mechanism of promoter activation in thymocytes from mice containing or lacking Ebeta. We found that pDbeta1 shows a complex profile of transcription factor and chromatin remodeling complex occupancy even at Ebeta(-) alleles. The presence of Ebeta, however, results in a few specific changes in factor occupancy at the promoter. These differences correlate with localized alterations in histone modifications and in the recruitment of the basal transcriptional machinery. In addition, Ebeta is also bound by CBP and Pol II, suggesting a mechanism for delivery of a holoenzyme complex to the pDbeta1 promoter. These results illustrate a specialized, long-range function of an enhancer in the hierarchical events that regulate assembly of a cell type-specific promoter.

Defining worldwide human genetic variation is a critical step to reveal how genome plasticity contributes to disease. Yet, there is currently no metric to assess the representativeness and completeness of current and widely used data on genetic variation. We show here that Human Leukocyte Antigen (HLA) genes can serve as such metric as they are both the most polymorphic and the most studied genetic system. As a test case, we investigated the 1,000 Genomes Project panel. Using high-accuracy in silico HLA typing, we find that over 20% of the common HLA variants and over 70% of the rare HLA variants are missing in this reference panel for worldwide genetic variation, due to undersampling and incomplete geographical coverage, in particular in Oceania and West Asia. Because common and rare variants both contribute to disease, this study thus illustrates how HLA diversity can detect and help fix incomplete sampling and hence accelerate efforts to draw a comprehensive overview of the genetic variation that is relevant to health and disease.