MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

Abstract

Recognition of short linear motifs (SLiMs) or peptides by proteins is an important component of many cellular processes. However, due to limited and degenerate binding motifs, prediction of cellular targets is challenging. In addition, many of these interactions are transient and of relatively low affinity. Here, we focus on one of the largest families of SLiM-binding domains in the human proteome, the PDZ domain. These domains bind the extreme C-terminus of target proteins, and are involved in many signaling and trafficking pathways. To predict endogenous targets of PDZ domains, we developed MotifAnalyzer-PDZ, a program that filters and compares all motif-satisfying sequences in any publicly available proteome. This approach enables us to determine possible PDZ binding targets in humans and other organisms. Using this program, we predicted and biochemically tested novel human PDZ targets by looking for strong sequence conservation in evolution. We also identified three C-terminal sequences in choanoflagellates that bind a choanoflagellate PDZ domain, the Monsiga brevicollis SHANK1 PDZ domain (mbSHANK1), with endogenously-relevant affinities, despite a lack of conservation with the targets of a homologous human PDZ domain, SHANK1. All three are predicted to be signaling proteins, with strong sequence homology to cytosolic and receptor tyrosine kinases. Finally, we analyzed and compared the positional amino acid enrichments in PDZ motifsatisfying sequences from over a dozen organisms. Overall, MotifAnalyzer-PDZ is a versatile program to investigate potential PDZ interactions. This proof-ofconcept work is poised to enable similar types of analyses for other SLiM-binding domains (e.g., MotifAnalyzer-Kinase). MotifAnalyzer-PDZ is available at

The evolution of non-motif selectivity determinants inMonosiga brevicollisPDZ domains

“…Our experimental structure, however, reveals that neither of the arginine residues in the vicinity are interacting with the P -3 Glu of GIRK3. It is unclear from our mbSHANK1:GIRK3 structure what may account for this slight difference in affinity, however the crystal structure confirms that the peptide-binding cleft is conserved as we previously reported, with the exception of the previously-described residues near the P -3 peptide position (46). In addition, the sequence and length of the bB-bC loop, which directly interacts with peptides in our structure, varies quite dramatically: The N-terminal residues of the 11-residue loop for mbSHANK1 PDZ are THSAAEQT, while for human SHANK1 PDZ, an 18-residue loop, they are KAQTPIEE.…”

“…The crystal structure of mbSHANK1 bound to F*-GIRK3 is structurally very similar to our previously determined homology model (46). The overall RMSD of these two structures is 0.668 Å over 276 main chain atoms, with the largest discrepancies occurring in the flexible bB-bC loop (Fig.…”

“…Our results reveal that overall, peptides which bind human SNX27 PDZ with relatively high affinity also bind mbSNX27 PDZ very strongly. We measure similar results with human SHANK1 and mbSHANK1 PDZ domains (46). However, in all cases, the exact order of highest to lowest affinity peptides is distinct, perhaps reflective of single substitutions in the peptide binding cleft.…”

“…Experimental protocols were based on previously described methods, and are described in more detail in the Experimental Procedures and Supporting Information (22,(46)(47)(48).…”

“…We previously compared binding affinities for another M. brevicollis PDZ domain, that of mbSHANK1 (UniProt ID: A9V7E4_MONBE), a protein that is homologous to human SHANK1 (46). In this work, we also created a homology model of mbSHANK1 PDZ using SwissModel and predicted stereochemical differences in the peptide binding pockets between these two proteins, specifically in those residues that interact with the P -3 position (46). Here, we expand that investigation by presenting the crystal structure of mbSHANK1 PDZ ( Fig.…”