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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

Molecular paints block trypsin cleavage sites.(a) Amino acids highlighted in red are consensus trypsin cleavage sites of CA II that were identified by reverse-phase liquid chromatography nanospray tandem MS in the absence of molecular paint (unpainted). Molecular paints (RBB, AO50, R49 and CR) blocked all (100%) consensus trypsin cleavage sites (painted, indicated by a blue X). CA was chosen because it contains 80% of the trypsin cleavage consensus sites that represent all the variations of the amino acids at the carboxy-side of the arginine and lysine. To further confirm that all the possible trypsin cleavage consensus domain were conserved as binding sites for the molecular paints, we conducted similar experiments for aprotinin and albumin in addition to the IL1β-IL1RI-IL1RAcP complex that documented full coverage of all known trypsin cleavage consensus sites for any permissible amino acid. Trypsin cleavage sites are the staple of mass spec sequencing because they mark the protein polypeptide chain at the highest frequency (resolution) compared with other protease cleavage sites40, and they are preferentially distributed on protein surfaces, and in or near previously identified solvent-excluded hot spots3. (b) 3D representation of crystallography structure of CA (PDB no. 1V9E). Surface trypsin cleavage sites are represented in magenta, (c) Yellow crosses indicate trypsin cleavage sites that were blocked by the four molecular paints used (RBB, AO50, R49 and CR). (d) Example representation of AO50 blocking Arg250 on the surface of CA as predicted by docking calculations (SwissDock53, PDB no. 1V9E, ZINC25693528).
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f4: Molecular paints block trypsin cleavage sites.(a) Amino acids highlighted in red are consensus trypsin cleavage sites of CA II that were identified by reverse-phase liquid chromatography nanospray tandem MS in the absence of molecular paint (unpainted). Molecular paints (RBB, AO50, R49 and CR) blocked all (100%) consensus trypsin cleavage sites (painted, indicated by a blue X). CA was chosen because it contains 80% of the trypsin cleavage consensus sites that represent all the variations of the amino acids at the carboxy-side of the arginine and lysine. To further confirm that all the possible trypsin cleavage consensus domain were conserved as binding sites for the molecular paints, we conducted similar experiments for aprotinin and albumin in addition to the IL1β-IL1RI-IL1RAcP complex that documented full coverage of all known trypsin cleavage consensus sites for any permissible amino acid. Trypsin cleavage sites are the staple of mass spec sequencing because they mark the protein polypeptide chain at the highest frequency (resolution) compared with other protease cleavage sites40, and they are preferentially distributed on protein surfaces, and in or near previously identified solvent-excluded hot spots3. (b) 3D representation of crystallography structure of CA (PDB no. 1V9E). Surface trypsin cleavage sites are represented in magenta, (c) Yellow crosses indicate trypsin cleavage sites that were blocked by the four molecular paints used (RBB, AO50, R49 and CR). (d) Example representation of AO50 blocking Arg250 on the surface of CA as predicted by docking calculations (SwissDock53, PDB no. 1V9E, ZINC25693528).

Mentions: We identified a panel of small, synthetic aryl hydrocarbon containing organic dyes (Fig. 1; Supplementary Figs 1 and 2; Supplementary Table 1), from a large number of candidate molecules (Supplementary Table 2), that bind to proteins as molecular paints. Paint chemistries were selected because they have extremely rapid on-rates (units: M−1 s−1) and very slow off-rates (<10−5 s−1, Supplementary Table 1) that are 10–100 times higher than most protein–protein interactions1920 (Fig. 2, using at least 10-fold molar excess of dye paint will coat 83% of low-affinity transient interactions that have been characterized21, Supplementary Fig. 3). Paint chemistries remain bound following protein dissociation or denaturation with 2 M urea (Fig. 3; Supplementary Table 1), and bind to multiple sites on the exposed protein surface to achieve complete masking of all the trypsin cleavage sites (Fig. 4; Supplementary Fig. 4).


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

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

Molecular paints block trypsin cleavage sites.(a) Amino acids highlighted in red are consensus trypsin cleavage sites of CA II that were identified by reverse-phase liquid chromatography nanospray tandem MS in the absence of molecular paint (unpainted). Molecular paints (RBB, AO50, R49 and CR) blocked all (100%) consensus trypsin cleavage sites (painted, indicated by a blue X). CA was chosen because it contains 80% of the trypsin cleavage consensus sites that represent all the variations of the amino acids at the carboxy-side of the arginine and lysine. To further confirm that all the possible trypsin cleavage consensus domain were conserved as binding sites for the molecular paints, we conducted similar experiments for aprotinin and albumin in addition to the IL1β-IL1RI-IL1RAcP complex that documented full coverage of all known trypsin cleavage consensus sites for any permissible amino acid. Trypsin cleavage sites are the staple of mass spec sequencing because they mark the protein polypeptide chain at the highest frequency (resolution) compared with other protease cleavage sites40, and they are preferentially distributed on protein surfaces, and in or near previously identified solvent-excluded hot spots3. (b) 3D representation of crystallography structure of CA (PDB no. 1V9E). Surface trypsin cleavage sites are represented in magenta, (c) Yellow crosses indicate trypsin cleavage sites that were blocked by the four molecular paints used (RBB, AO50, R49 and CR). (d) Example representation of AO50 blocking Arg250 on the surface of CA as predicted by docking calculations (SwissDock53, PDB no. 1V9E, ZINC25693528).
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Related In: Results  -  Collection

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f4: Molecular paints block trypsin cleavage sites.(a) Amino acids highlighted in red are consensus trypsin cleavage sites of CA II that were identified by reverse-phase liquid chromatography nanospray tandem MS in the absence of molecular paint (unpainted). Molecular paints (RBB, AO50, R49 and CR) blocked all (100%) consensus trypsin cleavage sites (painted, indicated by a blue X). CA was chosen because it contains 80% of the trypsin cleavage consensus sites that represent all the variations of the amino acids at the carboxy-side of the arginine and lysine. To further confirm that all the possible trypsin cleavage consensus domain were conserved as binding sites for the molecular paints, we conducted similar experiments for aprotinin and albumin in addition to the IL1β-IL1RI-IL1RAcP complex that documented full coverage of all known trypsin cleavage consensus sites for any permissible amino acid. Trypsin cleavage sites are the staple of mass spec sequencing because they mark the protein polypeptide chain at the highest frequency (resolution) compared with other protease cleavage sites40, and they are preferentially distributed on protein surfaces, and in or near previously identified solvent-excluded hot spots3. (b) 3D representation of crystallography structure of CA (PDB no. 1V9E). Surface trypsin cleavage sites are represented in magenta, (c) Yellow crosses indicate trypsin cleavage sites that were blocked by the four molecular paints used (RBB, AO50, R49 and CR). (d) Example representation of AO50 blocking Arg250 on the surface of CA as predicted by docking calculations (SwissDock53, PDB no. 1V9E, ZINC25693528).
Mentions: We identified a panel of small, synthetic aryl hydrocarbon containing organic dyes (Fig. 1; Supplementary Figs 1 and 2; Supplementary Table 1), from a large number of candidate molecules (Supplementary Table 2), that bind to proteins as molecular paints. Paint chemistries were selected because they have extremely rapid on-rates (units: M−1 s−1) and very slow off-rates (<10−5 s−1, Supplementary Table 1) that are 10–100 times higher than most protein–protein interactions1920 (Fig. 2, using at least 10-fold molar excess of dye paint will coat 83% of low-affinity transient interactions that have been characterized21, Supplementary Fig. 3). Paint chemistries remain bound following protein dissociation or denaturation with 2 M urea (Fig. 3; Supplementary Table 1), and bind to multiple sites on the exposed protein surface to achieve complete masking of all the trypsin cleavage sites (Fig. 4; Supplementary Fig. 4).

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