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Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Luchini A, Espina V, Liotta LA - Nat Commun (2014)

Bottom Line: The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface.We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP.We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling.

View Article: PubMed Central - PubMed

Affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.

ABSTRACT
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

No MeSH data available.


Related in: MedlinePlus

Protein painting reveals hot spots between IL1β, IL1RI and IL1RAcP.(a) Identified opposing contact points revealed by the method for the ligand (blue) bound to its receptor (green) before and after dissociation (Fisher exact test P-value<0.0003). Fisher exact test was applied in order to determine whether there was an enrichment of trypsin cleavage sites belonging to protein–protein interface regions among all the trypsin cleavage sites identified by the protein painting method. The total number of trypsin cleavage sites of the two proteins was 49 (26 in the protein–protein interface region). The total number of trypsin cleavage sites identified by the protein painting method was 17, 15 of which belonged to the interface (Fisher exact test P-value<0.0003). (b) IL1RAcP (pink) bound to the receptor (green) ligand (blue) complex. Sequences identified for each protein were found to be opposing and juxtaposed, as noted. The sequence labelled Arg286 (represented in black in the protein model) was used to generate a synthetic peptide antagonist and was used as an antigen for a mouse IgG mAb to Arg286 peptide. Protein painting correctly revealed key contact points with closest proximity obtained by X-ray crystallography and PDBePISA structural analysis software. The two closest interaction points in the three-way complex are IL1RAcP:Arg286–IL1β:Asp54, distance 2.49 Å and IL1RI:Lys298–IL1β:Ser52, distance 2.51 Å (PDB no. 4DEP). These contact points were correctly identified by protein painting in both protein partners. D1, D2 and D3 indicate the domains of IL1RI and IL1RAcP.
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f5: Protein painting reveals hot spots between IL1β, IL1RI and IL1RAcP.(a) Identified opposing contact points revealed by the method for the ligand (blue) bound to its receptor (green) before and after dissociation (Fisher exact test P-value<0.0003). Fisher exact test was applied in order to determine whether there was an enrichment of trypsin cleavage sites belonging to protein–protein interface regions among all the trypsin cleavage sites identified by the protein painting method. The total number of trypsin cleavage sites of the two proteins was 49 (26 in the protein–protein interface region). The total number of trypsin cleavage sites identified by the protein painting method was 17, 15 of which belonged to the interface (Fisher exact test P-value<0.0003). (b) IL1RAcP (pink) bound to the receptor (green) ligand (blue) complex. Sequences identified for each protein were found to be opposing and juxtaposed, as noted. The sequence labelled Arg286 (represented in black in the protein model) was used to generate a synthetic peptide antagonist and was used as an antigen for a mouse IgG mAb to Arg286 peptide. Protein painting correctly revealed key contact points with closest proximity obtained by X-ray crystallography and PDBePISA structural analysis software. The two closest interaction points in the three-way complex are IL1RAcP:Arg286–IL1β:Asp54, distance 2.49 Å and IL1RI:Lys298–IL1β:Ser52, distance 2.51 Å (PDB no. 4DEP). These contact points were correctly identified by protein painting in both protein partners. D1, D2 and D3 indicate the domains of IL1RI and IL1RAcP.

Mentions: We evaluated the power of protein painting by using it to study the multiple hot spots participating in the three-way interaction of IL1β ligand, its receptor IL1R1 and the accessory protein IL1RAcP (Fig. 5), a critically important drug target. Interleukin signalling requires the interaction of all three proteins101112. Aberrant function of this complex is involved in a variety of diseases, including cancer15, rheumatoid arthritis13, systemic lupus erythematosus16, inflammatory bowel disease16, systemic vasculitis16, neonatal bronchial dysplasia13, neurodegenerative conditions17 and inflammatory bone and cartilage destruction18.


Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Luchini A, Espina V, Liotta LA - Nat Commun (2014)

Protein painting reveals hot spots between IL1β, IL1RI and IL1RAcP.(a) Identified opposing contact points revealed by the method for the ligand (blue) bound to its receptor (green) before and after dissociation (Fisher exact test P-value<0.0003). Fisher exact test was applied in order to determine whether there was an enrichment of trypsin cleavage sites belonging to protein–protein interface regions among all the trypsin cleavage sites identified by the protein painting method. The total number of trypsin cleavage sites of the two proteins was 49 (26 in the protein–protein interface region). The total number of trypsin cleavage sites identified by the protein painting method was 17, 15 of which belonged to the interface (Fisher exact test P-value<0.0003). (b) IL1RAcP (pink) bound to the receptor (green) ligand (blue) complex. Sequences identified for each protein were found to be opposing and juxtaposed, as noted. The sequence labelled Arg286 (represented in black in the protein model) was used to generate a synthetic peptide antagonist and was used as an antigen for a mouse IgG mAb to Arg286 peptide. Protein painting correctly revealed key contact points with closest proximity obtained by X-ray crystallography and PDBePISA structural analysis software. The two closest interaction points in the three-way complex are IL1RAcP:Arg286–IL1β:Asp54, distance 2.49 Å and IL1RI:Lys298–IL1β:Ser52, distance 2.51 Å (PDB no. 4DEP). These contact points were correctly identified by protein painting in both protein partners. D1, D2 and D3 indicate the domains of IL1RI and IL1RAcP.
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Related In: Results  -  Collection

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f5: Protein painting reveals hot spots between IL1β, IL1RI and IL1RAcP.(a) Identified opposing contact points revealed by the method for the ligand (blue) bound to its receptor (green) before and after dissociation (Fisher exact test P-value<0.0003). Fisher exact test was applied in order to determine whether there was an enrichment of trypsin cleavage sites belonging to protein–protein interface regions among all the trypsin cleavage sites identified by the protein painting method. The total number of trypsin cleavage sites of the two proteins was 49 (26 in the protein–protein interface region). The total number of trypsin cleavage sites identified by the protein painting method was 17, 15 of which belonged to the interface (Fisher exact test P-value<0.0003). (b) IL1RAcP (pink) bound to the receptor (green) ligand (blue) complex. Sequences identified for each protein were found to be opposing and juxtaposed, as noted. The sequence labelled Arg286 (represented in black in the protein model) was used to generate a synthetic peptide antagonist and was used as an antigen for a mouse IgG mAb to Arg286 peptide. Protein painting correctly revealed key contact points with closest proximity obtained by X-ray crystallography and PDBePISA structural analysis software. The two closest interaction points in the three-way complex are IL1RAcP:Arg286–IL1β:Asp54, distance 2.49 Å and IL1RI:Lys298–IL1β:Ser52, distance 2.51 Å (PDB no. 4DEP). These contact points were correctly identified by protein painting in both protein partners. D1, D2 and D3 indicate the domains of IL1RI and IL1RAcP.
Mentions: We evaluated the power of protein painting by using it to study the multiple hot spots participating in the three-way interaction of IL1β ligand, its receptor IL1R1 and the accessory protein IL1RAcP (Fig. 5), a critically important drug target. Interleukin signalling requires the interaction of all three proteins101112. Aberrant function of this complex is involved in a variety of diseases, including cancer15, rheumatoid arthritis13, systemic lupus erythematosus16, inflammatory bowel disease16, systemic vasculitis16, neonatal bronchial dysplasia13, neurodegenerative conditions17 and inflammatory bone and cartilage destruction18.

Bottom Line: The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface.We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP.We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling.

View Article: PubMed Central - PubMed

Affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.

ABSTRACT
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

No MeSH data available.


Related in: MedlinePlus