The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/ Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between fulllength Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1-to 40-μM range. Furthermore, we identified several well-established host-virus proteinprotein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.T here are an estimated 650,000 protein-protein interactions in a human cell (1). These interactions are integral to cellular function and mediate signaling pathways that are often misregulated in cancer (2) and may be hijacked by viral proteins (3). Commonly, signaling pathways involve moderate affinity interactions between modular domains and short linear motifs (SLiMs; conserved 2-to 10-aa stretches in disordered regions) (4) that are difficult to capture using high-throughput methods, such as yeast two-hybrid (Y2H) or affinity-purification mass spectrometry (AP/MS) but can be identified using peptide arrays, splitprotein systems (5, 6), or peptide-phage display (7-10). A major limitation of peptide arrays is coverage, because the number of potential binding peptides in the proteome is orders of magnitude larger than what can be printed on an array. Conventional phage libraries display combinatorially generated peptide sequences that can identify biophysically optimal ligands of modular domains but this approach can exhibit a hydrophobic bias and may not be ideal for detecting natural binders (11). Thus, there is a need for alternative approaches for identification of relevant domain-SLiMs interactions.Here, we report an appr...

SignificanceUsing D-amino acids as the building blocks for bioactive peptides can dramatically increase their potency. However, simply swapping regular levorotary amino acids for dextrorotary (D)-amino acids alters the peptide surface topology and function is lost. Current methods to overcome this are not generally applicable and exclude the majority of therapeutic targets. By creating a mirror image of all 111,867 protein structures in the Protein Data Bank (PDB), we convert this repository into a D-peptide database with 2.8 million D-peptide structures. This D-PDB can be searched to find therapeutically active topologies, demonstrated here by the discovery of D-peptide GLP1R and PTH1R agonists. Evaluation of D-PDB coverage suggests that it holds candidates for most therapeutic targets and, thus, potentially contains hundreds of potent drug leads.

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 lM through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using fulllength proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PDderived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.

Neurons have an important role in retinal vascular development. Here we show that the G protein-coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1, previously known as Par2) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation interfere with Vegfa but not Ang1 expression. Complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.

Blocking the association between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) is an attractive therapeutic approach to prevent the virus from entering human cells. While antibodies and other modalities have been developed to this end, d -amino acid peptides offer unique advantages, including serum stability, low immunogenicity, and low cost of production. Here, we designed potent novel D-peptide inhibitors that mimic the ACE2 α1-binding helix by searching a mirror-image version of the PDB. The two best designs bound the RBD with affinities of 29 and 31 nM and blocked the infection of Vero cells by SARS-CoV-2 with IC 50 values of 5.76 and 6.56 μM, respectively. Notably, both D-peptides neutralized with a similar potency the infection of two variants of concern: B.1.1.7 and B.1.351 in vitro . These potent D-peptide inhibitors are promising lead candidates for developing SARS-CoV-2 prophylactic or therapeutic treatments.