Identifying Small‐Molecule Modulators of Protein‐Protein Interactions

Alexander R. Horswill1, Stephen J. Benkovic2

1 University of Iowa, Iowa City, Iowa, 2 The Pennsylvania State University, University Park, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.15
DOI:  10.1002/0471140864.ps1915s46
Online Posting Date:  December, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit outlines methods for identifying cyclic peptides that inhibit protein‐protein interactions. Proteins of interest are cloned into a two‐hybrid system engineered to operate in reverse, allowing the disruption of a protein complex to be coupled to cell growth. Cyclic peptide libraries are generated using an intein‐based plasmid construct, and the cyclized sequence is randomized using a PCR procedure. By transforming plasmid libraries into host cells containing the two‐hybrid fusions, cyclic peptide inhibitors can be identified by growing the cells under the appropriate selective conditions. A detailed procedure for performing the genetic selection and identifying false positives is provided. Methods for building the two‐hybrid protein fusions and optimizing media conditions, as well as an additional protocol for constructing cyclic peptide libraries are also provided.

Keywords: small‐molecule; protein‐protein; cyclic peptide; two‐hybrid; inhibitor

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Identification of Small‐Molecule Inhibitors of In Vivo Protein‐Protein Interactions by Genetic Selection
  • Support Protocol 1: Construction and Optimization of a Strain for Inhibitor Selection
  • Support Protocol 2: Construction of Cyclic Peptide Libraries
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Identification of Small‐Molecule Inhibitors of In Vivo Protein‐Protein Interactions by Genetic Selection

  Materials
  • Selection medium (see recipe)
  • Luria‐Bertani (LB) agar plates ( appendix 4A) supplemented with 25 µg/ml chloramphenicol
  • E. coli strain SNS‐126 containing the two‐hybrid reporter and protein domains of interest (or control protein) fused to the bacteriophage repressor DNA‐binding domains ( protocol 2)
  • 50 to 200 ng cyclic peptide library ( protocol 3)
  • SOC broth (see recipe)
  • 1× MMA: diluted from 5× minimal medium A ( appendix 4A)
  • LB broth ( appendix 4A) with and without 25 µg/ml chloramphenicol
  • 200 mM IPTG
  • Oligonucleotide that hybridizes to the intein fragment (in this case, pAR‐NdeI, 5′‐ GGAATTCCATATGGTTAAAGTTATCGGTCGTCGT‐3′)
  • 245 × 245–mm culture plates (Nunc)
  • 100 × 15–mm petri dishes
  • Electroporator and cuvettes
  • 18 × 100–mm culture tubes
  • Toothpicks, sterile
  • Inoculating loops, sterile
  • Plasmid preparation kit, optional (e.g., QIAquick Spin Miniprep Kit; Qiagen)
  • 96‐well microtiter plates
  • Multichannel pipettor
  • DNA sequencing facility
  • Additional reagents and equipment for growing bacteria ( appendix 4B), preparing electrocompetent cells for electroporation (Seidman et al., ), and purifying plasmid DNA ( appendix 4C)
NOTE: All reagents used with E. coli cells must be sterile.

Support Protocol 1: Construction and Optimization of a Strain for Inhibitor Selection

  Materials
  • Oligonucleotides (specifically designed)
  • Plasmid pTHCP16 (Fig. A, for making homodimeric fusions) or pTHCP14 (Fig. C, for making heterodimeric fusions)
  • DNA template (gene of interest)
  • 2.5 U/ml proofreading PCR polymerase (e.g., Deep Vent, New England Biolabs; Pfu, Stratagene)
  • 0.1 M EDTA, pH 8.0
  • 10% SDS
  • 2.5 mg/ml proteinase K
  • 3 M sodium acetate, pH 5.2
  • 70% (v/v) and 100% ethanol
  • PCR purification kit (e.g., QIAquick, Qiagen; optional)
  • DNA purification kit (e.g., QIAquick Spin Miniprep Kit, Qiagen; optional)
  • Restriction enzymes (specific to the cloning procedure)
  • Gel extraction kit (e.g., QIAquick gel extraction kit, Qiagen; optional)
  • T4 DNA ligase (New England Biolabs)
  • E. coli competent cells (e.g., DH5α): prepared ( appendix 4D) or purchased from commercial suppliers
  • LB agar plates ( appendix 4A) supplemented with 100 µg/ml ampicillin and with and without IPTG (0 to 1 mM)
  • E. coli strain SNS‐118 (homodimeric reporter) or SNS‐126 (heterodimeric reporter)
  • CRIM plasmids (E. coli Genetic Stock Center, Yale), optional
  • Z buffer (see recipe)
  • Chloroform
  • 0.1% (w/v) SDS
  • 4 mg/ml o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG): prepared fresh
  • 1 M sodium bicarbonate (Na 2CO 3)
  • ¾ in. sterile, optical glass culture tubes, optional
  • 13 × 100–mm glass tubes (disposable)
  • Additional reagents and equipment for growing bacteria ( appendix 4B), performing PCR ( appendix 4J), separating DNA by agarose gel electrophoresis ( appendix 4F), purifying plasmid DNA ( appendix 4C), quantitating DNA ( appendix 4K)
NOTE: For more information about preparing, analyzing, and cloning DNA see appendix 44.

Support Protocol 2: Construction of Cyclic Peptide Libraries

  Materials
  • LB broth ( appendix 4A) supplemented with 30 µg/ml chloramphenicol
  • E. coli culture containing plasmid pARCBD‐p (Scott et al., ), or another compatible plasmid
  • DNA purification kit (e.g., QIAquick Spin Miniprep Kit, Qiagen; optional)
  • 2.5 U/µl proofreading PCR polymerase (e.g., Deep Vent; New England Biolabs) and 10× buffer
  • 2 mM (each) dNTP mix
  • Oligonucleotide primers
    • C+5 (5′‐GGAATTCGCCAATGGGGCGATCGCCCACAATTGTNNSNNSNNSNNSNNSTGCTTAAGTTTTGGC‐3′)
    • CBD‐reverse (5′‐GGAATTCAAGCTTTCATTGAAGCTGCCACAAGG‐3′)
    • zipper (5′‐GGAATTCGCCAATGGGGCGATCGCC‐3′)
  • PCR purification kit, optional (e.g., QIAquick,Qiagen)
  • 100 mM EDTA, pH 8
  • 10% (w/v) SDS
  • 2.5 mg/ml proteinase K
  • Restriction enzymes: BglI and HindIII (e.g., New England Biolabs)
  • Phosphatase (e.g., shrimp alkaline phosphatase; United States Biochemical)
  • Gel extraction kit (e.g., QIAquick gel extraction kit, Qiagen)
  • T4 DNA ligase (e.g., Promega)
  • 3 M sodium acetate, pH 5.2
  • 100% ethanol
  • Thermal cycler
  • 0.05 micron VM nitrocellulose membranes (Millipore), optional
  • 250‐ml beaker, optional
  • Additional reagents and equipment for purifying plasmid DNA ( appendix 4C), purifying DNA ( appendix 4E), performing agarose gel electrophoresis ( appendix 4F), and quantitating DNA by spectroscopy ( appendix 4K)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abel‐Santos, E., Scott, C.P. and Benkovic, S.J. 2003. Use of inteins for the in vivo production of stable cyclic peptide libraries in E. coli. Methods Mol. Biol. 205:281‐294.
   Brennan, M.B. and Struhl, K. 1980. Mechanisms of increasing expression of a yeast gene in Escherichia coli. J. Mol. Biol. 136:333‐338.
   Clackson, T. and Wells, J.A. 1995. A hot spot of binding energy in a hormone‐receptor interface. Science 267:383‐386.
   Cochran, A.G. 2000. Antagonists of protein‐protein interactions. Chem. Biol. 7:R85‐94.
   Di Lallo, G., Castagnoli, L., Ghelardini, P., and Paolozzi, L. 2001. A two‐hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli. Microbiology 147:1651‐1656.
   Geyer, C.R., Colman‐Lerner, A., and Brent, R. 1999. “Mutagenesis” by peptide aptamers identifies genetic network members and pathway connections. Proc. Natl. Acad. Sci. U.S.A. 96:8567‐8572.
   Goryshin, I.Y., Jendrisak, J., Hoffman, L.M., Meis, R., and Reznikoff, W.S. 2000. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat. Biotechnol. 18:97‐100.
   Grigoriev, A. 2003. On the number of protein‐protein interactions in the yeast proteome. Nucleic Acids Res. 31:4157‐4161.
   Haldimann, A. and Wanner, B.L. 2001. Conditional‐replication, integration, excision, and retrieval plasmid‐host systems for gene structure‐function studies of bacteria. J. Bacteriol. 183:6384‐6393.
   Hirschmann, R., Hynes, J., Jr., Cichy‐Knight, M.A., van Rijn, R.D., Sprengler, P.A., Spoors, P.G., Shakespeare, W.C., Pietranico‐Cole, S., Barbosa, J., Liu, J., Yao, W., Rohrer, S., and Smith, A.B., 3rd. 1998. Modulation of receptor and receptor subtype affinities using diastereomeric and enantiomeric monosaccharide scaffolds as a means to structural and biological diversity. A new route to ether synthesis. J. Med. Chem. 41:1382‐1391.
   Hoppe‐Seyler, F., Crnkovic‐Mertens, I., Denk, C., Fitscher, B.A., Klevenz, B., Tomai, E., and Butz, K. 2001. Peptide aptamers: new tools to study protein interactions. J. Steroid. Biochem. Mol. Biol. 78:105‐111.
   Horswill, A.R., Savinov, S.N., and Benkovic, S.J. 2004. A systematic method for identifying small‐molecule modulators of protein‐protein interactions. Proc. Natl. Acad. Sci. U.S.A 101:15591‐15596.
   Huang, J. and Schreiber, S.L. 1997. A yeast genetic system for selecting small molecule inhibitors of protein‐protein interactions in nanodroplets. Proc. Natl. Acad. Sci. U.S.A. 94:13396‐13401.
   Joung, J.K., Ramm, E.I., and Pabo, C.O. 2000. A bacterial two‐hybrid selection system for studying protein‐DNA and protein‐protein interactions. Proc. Natl. Acad. Sci. U.S.A. 97:7382‐7387.
   Khlebnikov, A., Datsenko, K.A., Skaug, T., Wanner, B.L., and Keasling, J.D. 2001. Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low‐affinity high‐capacity AraE transporter. Microbiology 147:3241‐3247.
   Ladant, D. and Karimova, G. 2000. Genetic systems for analyzing protein‐protein interactions in bacteria. Res. Microbiol. 151:711‐720.
   Leanna, C.A. and Hannink, M. 1996. The reverse two‐hybrid system: a genetic scheme for selection against specific protein/protein interactions. Nucleic Acids Res. 24:3341‐3347.
   Marcotte, E.M., Pellegrini, M., Ng, H.L., Rice, D.W., Yeates, T.O., and Eisenberg, D. 1999. Detecting protein function and protein‐protein interactions from genome sequences. Science 285:751‐753.
   Miller, J. 1992. Procedures for working with lac. In A Short Course in Bacterial Genetics, pp. 71‐80. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Naumann, T.A., Savinov, S.N., and Benkovic, S.J. 2005. Engineering an affinity tag for genetically encoded cyclic peptides. Biotechnol. Bioeng. 92:820‐830.
   Norman, T.C., Smith, D.L., Sorger, P.K., Drees, B.L., O'Rourke, S.M., Hughes, T.R., Roberts, C.J. Friend, S.H., Fields, S., and Murray, A.W. 1999. Genetic selection of peptide inhibitors of biological pathways. Science 285:591‐595.
   Park, S.H. and Raines, R.T. 2000. Genetic selection for dissociative inhibitors of designated protein‐protein interactions. Nat. Biotechnol. 18:847‐851.
   Ramani, A.K., Bunescu, R.C., Mooney, R.J., and Marcotte, E.M. 2005. Consolidating the set of known human protein‐protein interactions in preparation for large‐scale mapping of the human interactome. Genome Biol. 6:R40.
   Scott, C.P., Abel‐Santos, E., Wall, M., Wahnon, D.C., and Benkovic, S.J. 1999. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. U.S.A. 96:13638‐13643.
   Scott, C.P., Abel‐Santos, E., Jones, A.D., and Benkovic, S.J. 2001. Structural requirements for the biosynthesis of backbone cyclic peptide libraries. Chem. Biol. 8:801‐815.
   Seidman, C.E., Struhl, K., and Sheen, J. 2002. Introduction of plasmid DNA into cells. In Short Protocols in Molecular Biology, 5th ed. (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1‐29 to 1‐31. John Wiley & Sons, New York.
   Serebriiskii, I.G., Mitina, O., Pugacheva, E.N., Benevolenskaya, E., Kotova, E., Toby, G.G., Khazak, V., Kaelin, W.G., Chernoff, J., and Golemis, E.A. 2002. Detection of peptides, proteins, and drugs that selectively interact with protein targets. Genome Res. 12:1785‐1791.
   Tavassoli, A. and Benkovic, S.J. 2005. Genetically selected cyclic‐peptide inhibitors of AICAR transformylase homodimerization. Angew. Chem. Int. Ed. Engl. 44:2760‐2763.
   Toogood, P.L. 2002. Inhibition of protein‐protein association by small molecules: approaches and progress. J. Med. Chem. 45:1543‐1558.
   Vidal, M., Brachmann, R.K., Fattaey, A., Harlow, E., and Boeke, J.D. 1996. Reverse two‐hybrid and one‐hybrid systems to detect dissociation of protein‐protein and DNA‐protein interactions. Proc. Natl. Acad. Sci. U.S.A. 93:10315‐10320.
   Vidal, M. and Endoh, H. 1999. Prospects for drug screening using the reverse two‐hybrid system. Trends Biotechnol. 17:374‐381.
   Walker, J.R., Roth, J.R., and Altman, E. 2001. An in vivo study of novel bioactive peptides that inhibit the growth of Escherichia coli. J. Pept. Res. 58:380‐388.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library