A multi-reporter bacterial 2-hybrid assay for the fast, simple, and dynamic assay of PDZ domain – peptide interactions

The accurate determination of protein-protein interactions is an important goal of molecular biology and much progress has been made with modern methods emerging in the past decade. However, current methods have limitations, including scale and restriction to high affinity interactions. This has limited our understanding of the large subset of protein-motif interactions. Here we describe a modified bacterial-hybrid assay that employs a combined selectable and scalable reporter system that allows the screen of large protein-peptide libraries and their sort by relative strength. We have applied this tool to characterize a set of human and E. coli PDZ domains. Our results are consistent with prior characterization of these proteins and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.

[1]  Katja Luck,et al.  Phage display can select over-hydrophobic sequences that may impair prediction of natural domain-peptide interactions , 2011, Bioinform..

[2]  M. Noyes,et al.  A systematic characterization of factors that regulate Drosophila segmentation via a bacterial one-hybrid system , 2008, Nucleic acids research.

[3]  M. Vidal,et al.  Protein-protein interactions and networks: forward and reverse edgetics. , 2011, Methods in molecular biology.

[4]  Tony Pawson,et al.  Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes , 2014, Proceedings of the National Academy of Sciences.

[5]  Renaud Vincentelli,et al.  Quantifying domain-ligand affinities and specificities by high-throughput holdup assay , 2015, Nature Methods.

[6]  T. Baker,et al.  Steric clashes with bound OMP peptides activate the DegS stress-response protease , 2015, Proceedings of the National Academy of Sciences.

[7]  Sachdev S Sidhu,et al.  Origins of PDZ Domain Ligand Specificity , 2003, The Journal of Biological Chemistry.

[8]  Alan Bridge,et al.  New and continuing developments at PROSITE , 2012, Nucleic Acids Res..

[9]  R. Sauer,et al.  The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation , 1996, Journal of bacteriology.

[10]  J. Snider,et al.  Detecting Membrane Protein‐protein Interactions Using the Mammalian Membrane Two‐hybrid (MaMTH) Assay , 2017, Current protocols in chemical biology.

[11]  Edward L. Huttlin,et al.  The BioPlex Network: A Systematic Exploration of the Human Interactome , 2015, Cell.

[12]  Bridget E. Begg,et al.  A Proteome-Scale Map of the Human Interactome Network , 2014, Cell.

[13]  H. Saibil,et al.  Structural basis for the regulated protease and chaperone function of DegP , 2008, Nature.

[14]  G. Stormo,et al.  Analysis of Homeodomain Specificities Allows the Family-wide Prediction of Preferred Recognition Sites , 2008, Cell.

[15]  Aidan Budd,et al.  Short linear motifs: ubiquitous and functionally diverse protein interaction modules directing cell regulation. , 2014, Chemical reviews.

[16]  Tony Pawson,et al.  The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells , 2014, Nature Methods.

[17]  Todd H. Oakley,et al.  Evolutionary expansion and specialization of the PDZ domains. , 2010, Molecular biology and evolution.

[18]  R. Sauer,et al.  Allosteric Activation of DegS, a Stress Sensor PDZ Protease , 2007, Cell.

[19]  A. Emili,et al.  A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome. , 2017, Molecular cell.

[20]  H. Lehrach,et al.  A Human Protein-Protein Interaction Network: A Resource for Annotating the Proteome , 2005, Cell.

[21]  Benjamin L. Oakes,et al.  Deep sequencing of large library selections allows computational discovery of diverse sets of zinc fingers that bind common targets , 2013, Nucleic acids research.

[22]  M. Brodsky,et al.  A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors , 2005, Nature Biotechnology.

[23]  P. Güntert,et al.  Proteome‐wide analysis of phospho‐regulated PDZ domain interactions , 2018, Molecular systems biology.

[24]  Benjamin L. Oakes,et al.  A systematic survey of the Cys2His2 zinc finger DNA-binding landscape , 2015, Nucleic acids research.

[25]  M. Noyes.  Analysis of specific protein-DNA interactions by bacterial one-hybrid assay. , 2012, Methods in molecular biology.

[26]  Hyeong Jun An,et al.  Estimating the size of the human interactome , 2008, Proceedings of the National Academy of Sciences.

[27]  Gabor T. Marth,et al.  A global reference for human genetic variation , 2015, Nature.

[28]  Kevin Y. Yip,et al.  Identification of specificity determining residues in peptide recognition domains using an information theoretic approach applied to large-scale binding maps , 2011, BMC Biology.

[29]  R. Ozawa,et al.  A comprehensive two-hybrid analysis to explore the yeast protein interactome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  S. Galande,et al.  PDZ Domains in Microorganisms: Link Between Stress Response and Protein Synthesis , 2017, bioRxiv.

[31]  Bartek Wilczynski,et al.  Optimally choosing PWM motif databases and sequence scanning approaches based on ChIP-seq data , 2015, BMC Bioinformatics.

[32]  T. Pawson,et al.  Assembly of Cell Regulatory Systems Through Protein Interaction Domains , 2003, Science.

[33]  Jiunn R Chen,et al.  PDZ Domain Binding Selectivity Is Optimized Across the Mouse Proteome , 2007, Science.

[34]  Jie J. Zheng,et al.  ReviewPDZ domains and their binding partners : structure , specificity , and modification , 2010 .

[35]  L. Castagnoli,et al.  mentha: a resource for browsing integrated protein-interaction networks , 2013, Nature Methods.

[36]  S. L. Wong,et al.  Towards a proteome-scale map of the human protein–protein interaction network , 2005, Nature.

[37]  Prisca Boisguerin,et al.  Engineering peptide inhibitors to overcome PDZ binding promiscuity. , 2010, Angewandte Chemie.

[38]  P. Bork,et al.  Nutritional preferences of human gut bacteria reveal their metabolic idiosyncrasies , 2018, Nature Microbiology.

[39]  Kenny Q. Ye,et al.  An integrated map of genetic variation from 1,092 human genomes , 2012, Nature.

[40]  Katja Luck,et al.  The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains , 2012, FEBS letters.

[41]  James Y. Zou.  Analysis of protein-coding genetic variation in 60,706 humans , 2015, Nature.

[42]  Raffi Tonikian,et al.  Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries , 2007, Nature Protocols.

[43]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[44]  Chris Sander,et al.  A Specificity Map for the PDZ Domain Family , 2008, PLoS biology.

[45]  Norman E. Davey,et al.  The switches.ELM Resource: A Compendium of Conditional Regulatory Interaction Interfaces , 2013, Science Signaling.

[46]  Tim Clausen,et al.  Crystal Structure of the DegS Stress Sensor How a PDZ Domain Recognizes Misfolded Protein and Activates a Protease , 2004, Cell.

[47]  M. Vidal,et al.  Protein interaction mapping in C. elegans using proteins involved in vulval development. , 2000, Science.

[48]  Benjamin L. Oakes,et al.  Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing , 2016, Nature Communications.