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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 hidden native hot spots of protein interactions.(a) Paint molecules coat the surface of native protein complexes but cannot gain access to solvent-inaccessible protein–protein interface regions. Interleukin 1β receptor–ligand complex depicted with bound paint molecules to scale. (b) Trypsin cleavage is blocked by the presence of paint molecules that bind non-covalently near trypsin consensus sequences. Following dissociation of painted proteins the area of interaction remains unpainted and is susceptible to trypsin cleavage. Thus, trypsin cleavage peptides will be derived exclusively from unpainted interface areas.
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f1: Protein painting reveals hidden native hot spots of protein interactions.(a) Paint molecules coat the surface of native protein complexes but cannot gain access to solvent-inaccessible protein–protein interface regions. Interleukin 1β receptor–ligand complex depicted with bound paint molecules to scale. (b) Trypsin cleavage is blocked by the presence of paint molecules that bind non-covalently near trypsin consensus sequences. Following dissociation of painted proteins the area of interaction remains unpainted and is susceptible to trypsin cleavage. Thus, trypsin cleavage peptides will be derived exclusively from unpainted interface areas.

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)

Protein painting reveals hidden native hot spots of protein interactions.(a) Paint molecules coat the surface of native protein complexes but cannot gain access to solvent-inaccessible protein–protein interface regions. Interleukin 1β receptor–ligand complex depicted with bound paint molecules to scale. (b) Trypsin cleavage is blocked by the presence of paint molecules that bind non-covalently near trypsin consensus sequences. Following dissociation of painted proteins the area of interaction remains unpainted and is susceptible to trypsin cleavage. Thus, trypsin cleavage peptides will be derived exclusively from unpainted interface areas.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109009&req=5

f1: Protein painting reveals hidden native hot spots of protein interactions.(a) Paint molecules coat the surface of native protein complexes but cannot gain access to solvent-inaccessible protein–protein interface regions. Interleukin 1β receptor–ligand complex depicted with bound paint molecules to scale. (b) Trypsin cleavage is blocked by the presence of paint molecules that bind non-covalently near trypsin consensus sequences. Following dissociation of painted proteins the area of interaction remains unpainted and is susceptible to trypsin cleavage. Thus, trypsin cleavage peptides will be derived exclusively from unpainted interface areas.
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