In this study, we describe the first PDZ protein-ligand interaction network coupled with quantitative binding strength. Our network approaches elucidated how PDZ domains have diversified their binding partners in the organization of various signaling complexes from receptors to downstream signaling relays. Moreover, we showed that de novo evolution of PDZ domain-ligand interactions played an important role in the growth of PDZNet. These findings provide empirical evidence for a network evolution model that highlights the rewiring of interactions as a mechanism of functional innovation.
PDZNet provides information beyond just the state of interaction binding. First, PDZNet provides information regarding the binding interface. High-throughput experiments provided large-scale PPI information; however, the identification of which amino acids were used in the interactions has been difficult. The quantitative model of PDZ domain-ligand interactions provides sequence information on domains and linear motifs, enabling a deeper understanding of the mechanisms involved in their interactions. Second, PDZNet provides the binding strengths of the interactions. The quantitative binding strengths of PDZ domain-ligand interactions enable us to understand the competition among interaction partners for switching between signaling flows.
The multispecificity of PDZ domain-ligand interactions has unique advantages in the evolution of PDZ domain function in the cell signaling network. First, the multispecificity of PDZ domains contributes to the frequent rewiring of PDZ domain-ligand interactions and broadens the extent of recognizable sequences, thus increasing the chance that a protein gains a suitable sequence to interact with its partners. Indeed, we found that PDZ domain pockets prefer multiple amino acids for interactions. We analyzed amino acid preference patterns from the PWMs of human PDZ domains (Figure S8
) and found that the degeneracy of binding motifs facilitate the binding of different PDZ ligands to the same PDZ domain. This finding is consistent with those of a recent study that revealed the specificities of PDZ domains lie on a continuum 
. Second, the multispecificity of PDZ domains enables the combinatorial assembly of signaling complexes that control signaling processes. PDZ proteins interact with many signaling proteins and form preassembled complexes, which are important for the precision of information flow and the fidelity of cell signaling events 
. An interesting observation from our network approach is that a PDZ protein is connected to many ligands. These ligands may interact with a PDZ protein in a tissue-specific manner; the subsequent cell type-dependent expression of the PDZ ligands may lead to an alternative assembly of signaling complexes, thus enabling cell type-specific responses for extracellular signals. Indeed, we observed that the ligands of the SAP97 PDZ protein showed tissue-specific expression patterns, allowing the formation of tissue-specific cell signaling complexes (Figure S9
). Third, the multispecific interactions of PDZ domains may enhance the robustness of the signaling processes mediated by PDZ domains. The robustness of the cell signaling network is known to increase because several means often exist to achieve one function as the failure of one can be compensated by others 
. In PDZNet, PDZ domains tend to interact with a series of homologous proteins, particularly cell surface receptors. This interaction may ensure reliable transmission of signals mediated by PDZ proteins to the plasma membrane.
We found that almost one-third of human PDZ ligands obtained their PDZ-binding motifs via C-terminal sequence mutations, providing evolutionary advantages to the PDZ domain-mediated interactions. First, the formation of linear motifs is an efficient mechanism to increase the number of interactions. Emergence of short linear motifs rarely disrupts the protein structure and can be accompanied by few amino acid changes 
. Second, the de novo
evolution of interactions via sequence mutation provides an effective means for functional innovation. Gene duplication is known to have a limited role in the molecular innovation of biochemical function but facilitates the modularization of functional networks by specialization 
. In contrast, the de novo
evolution of interactions allows connections between evolutionarily unrelated functional modules, thus enabling the reconfiguration of the molecular system. For instance, gain of the PDZ domain-ligand interaction between the EXOC4 PDZ ligand and the SAP102 PDZ protein demonstrated an innovation by bridging two different functional modules. We examined species-specific functional annotations of PDZ ligands and found that yeast EXOC4 participates in vesicle transport with other exocyst complex members, but vertebrate EXOC4 regulates NMDAR transport to the postsynaptic membrane by interacting with the SAP102 PDZ domain 
. Third, when a PDZ protein gains ligands by sequence mutation, it avoids a loss of fitness caused by an increase in dosage. The de novo
evolution of PDZ domain-ligand interactions does not increase the copy number of the PDZ ligand genes, avoiding an unfavorable increase in protein concentration. In contrast, gain of interactions by duplication may cause a loss of fitness because proteins that contain linear motifs tend to be intrinsically disordered and dosage sensitive 
We were also interested in whether new PDZ domain interaction sites were acquired via C-terminal point mutations or DNA insertions. After careful observation of DNA modifications in newly acquired PDZ ligands, we found instances of both. For example, protein PBK of Macaca mulatta
acquired PDZ domain interaction motif “ETDV” via C-terminal point mutations in which a single nucleotide substitution (T→C) changed Ile to Thr and another mutation (C→T) changed the codon for Gln to a stop codon (Figure S10A
). On the other hand, EXOC4 acquired new PDZ domain interaction sites via DNA insertion in Oryzias latipes
). A large section of DNA inserted near the C-terminus of EXOC4 caused a frame shift mutation, which in turn became the PDZ domain-binding motif “ITTV.”
We found that the rewiring of PDZ domain-ligand interactions most frequently occurred between invertebrates and vertebrates. This massive rewiring may be connected to repeated rounds of whole-genome evolution in ancestral vertebrates. According to Ohno's model 
, when a gene is duplicated, mutations freely accumulate in the redundant duplicate copy under no selection. Therefore, the duplicate copy has a greater chance of developing new functions without altering existing functions. This evolutionary mechanism may facilitate network rewiring in early vertebrates.
We found that the components of PDZNet are largely associated with neurological diseases. We then asked whether we could identify mutations affecting PDZ-ligand binding, which causes genetic diseases. The disruption of the PDZ domain interaction between PICK1 and GluR7 is known to cause seizures, a chronic neurological disease 
. Mutations in the C-terminal sequence of GluR7 disrupted its PDZ domain interaction with PICK1. To examine whether our quantitative model can predict the effects of mutations in GluR7, we generated the PWM of the PICK PDZ domain and calculated the binding scores for both the wild-type and mutant forms of GluR7 (Figure S11
). We found that the wild type had a high binding score (5.98), and the mutant had a much lower binding score (−0.02). This example illustrates how our method can be applied to characterize genetic diseases that are caused by mutations affecting PDZ domain-ligand interactions.
An important issue of the present biological network study is its incompleteness 
. PDZNet has room for improvement regarding network coverage in two respects: shortage of nodes and links in the current network. To test whether the conclusions obtained in this work are sufficiently robust with regard to both, we constructed smaller random networks from PDZNet and repeated the analyses. In each trial, 20% of the proteins or interactions were randomly removed from PDZNet. We found that in all tests, the overall organization of the rescaled PDZNet remained largely unaltered, and the conclusions and the differences between the paralog fractions of the PDZ proteins and ligands were retained (Figures S12
), supporting the robustness of our findings to the future expansion of PDZ domain-ligand interactions.
Due to the incompleteness of the interactome networks, expansion of network coverage is of significant value. PDZ domain-ligand interactions were relatively difficult to detect using current experimental techniques because transient interactions are often lost during experimental washing steps. Furthermore, a PDZ domain-ligand interaction often depends on phosphorylation 
, so it can be missed when screening for protein interactions preformed in a single condition. Therefore, many PDZ domain-ligand interactions remain to be discovered. We anticipate that putative PDZ domain-ligand interactions with high-binding scores from PWMs, expression correlations, and similar phylogenetic profiles may be used to uncover novel interactions. Therefore, we provide a candidate list of PDZ domain-ligand interactions to assist in the discovery of novel PDZ domain-ligand interactions (sbi.postech.ac.kr/pdz).