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Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling.

Rizk SS, Kouadio JL, Szymborska A, Duguid EM, Mukherjee S, Zheng J, Clevenger CV, Kossiakoff AA - Cell Commun. Signal (2015)

Bottom Line: This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding.We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA. srizk@nd.edu.

ABSTRACT

Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response.

Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.

Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

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Related in: MedlinePlus

Crystal structure of the hPRL-R in complex with sAB A8. a sAB A8 (green) binds to the human PRL-R (red) opposite to the hormone binding site. b A close up of the interactions between the CDR loops of sAB A8 (green) and the hinge region between the two fibronectin (FN) domains of PRL-R (red). c Alignment of the FN2 domains of the PRL-R structure determined here (red) with the PRL-R structure (yellow) bound to PRL (grey) in the 2:1 complex (PDB code: 3NPZ), the side view (above) shows the twist between the two FN domains induced by sAB binding and the top view (below) shows a rotation of the FN1 domain relative to the FN2 domain in the sAB-bound structure. d Alignment of the sAB-bound PRL-R (red) with the hGH-bound PRL-R (green) and the PRL-bound PRL-R (yellow) using the FN2. The bottom panel is a top view of the FN1 of all three structures, with the sAB-bound receptor more resembling the hGH-bound conformation. e Inhibition of sAB binding by hGH or PRL using phage ELISA. Both hGH (left panel) and PRL (right panel) inhibit the interaction between phage-displayed sABs and hPRL-R in a concentration dependent manner. f IC50 values of the hGH or PRL inhibition of the interaction between sABs and hPRL-R calculated from e.
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Fig1: Crystal structure of the hPRL-R in complex with sAB A8. a sAB A8 (green) binds to the human PRL-R (red) opposite to the hormone binding site. b A close up of the interactions between the CDR loops of sAB A8 (green) and the hinge region between the two fibronectin (FN) domains of PRL-R (red). c Alignment of the FN2 domains of the PRL-R structure determined here (red) with the PRL-R structure (yellow) bound to PRL (grey) in the 2:1 complex (PDB code: 3NPZ), the side view (above) shows the twist between the two FN domains induced by sAB binding and the top view (below) shows a rotation of the FN1 domain relative to the FN2 domain in the sAB-bound structure. d Alignment of the sAB-bound PRL-R (red) with the hGH-bound PRL-R (green) and the PRL-bound PRL-R (yellow) using the FN2. The bottom panel is a top view of the FN1 of all three structures, with the sAB-bound receptor more resembling the hGH-bound conformation. e Inhibition of sAB binding by hGH or PRL using phage ELISA. Both hGH (left panel) and PRL (right panel) inhibit the interaction between phage-displayed sABs and hPRL-R in a concentration dependent manner. f IC50 values of the hGH or PRL inhibition of the interaction between sABs and hPRL-R calculated from e.

Mentions: To gain insight into the structural basis of the interactions between the sABs and hPRL-R, we determined the crystal structure of the receptor in complex with sAB A8. In the structure, the sAB binds to the opposite face of the ECD away from the hormone-binding site (Figure 1a). Interactions between the two molecules are mediated by side chain residues of the CDR loops of the sAB and the hinge region that connects the two tandem fibronectin (FN) domains of hPRL-R (Figure 1b). Alignment of the ECD FN2 domain in the context of the sAB-bound structure with the 2:1 PRLR: PRL structure [8] reveals that the sAB captures a hinge-bending conformation that results in a subtle, but significant rotation of FN1 relative to FN2 (Figure 1c).Figure 1


Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling.

Rizk SS, Kouadio JL, Szymborska A, Duguid EM, Mukherjee S, Zheng J, Clevenger CV, Kossiakoff AA - Cell Commun. Signal (2015)

Crystal structure of the hPRL-R in complex with sAB A8. a sAB A8 (green) binds to the human PRL-R (red) opposite to the hormone binding site. b A close up of the interactions between the CDR loops of sAB A8 (green) and the hinge region between the two fibronectin (FN) domains of PRL-R (red). c Alignment of the FN2 domains of the PRL-R structure determined here (red) with the PRL-R structure (yellow) bound to PRL (grey) in the 2:1 complex (PDB code: 3NPZ), the side view (above) shows the twist between the two FN domains induced by sAB binding and the top view (below) shows a rotation of the FN1 domain relative to the FN2 domain in the sAB-bound structure. d Alignment of the sAB-bound PRL-R (red) with the hGH-bound PRL-R (green) and the PRL-bound PRL-R (yellow) using the FN2. The bottom panel is a top view of the FN1 of all three structures, with the sAB-bound receptor more resembling the hGH-bound conformation. e Inhibition of sAB binding by hGH or PRL using phage ELISA. Both hGH (left panel) and PRL (right panel) inhibit the interaction between phage-displayed sABs and hPRL-R in a concentration dependent manner. f IC50 values of the hGH or PRL inhibition of the interaction between sABs and hPRL-R calculated from e.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4300558&req=5

Fig1: Crystal structure of the hPRL-R in complex with sAB A8. a sAB A8 (green) binds to the human PRL-R (red) opposite to the hormone binding site. b A close up of the interactions between the CDR loops of sAB A8 (green) and the hinge region between the two fibronectin (FN) domains of PRL-R (red). c Alignment of the FN2 domains of the PRL-R structure determined here (red) with the PRL-R structure (yellow) bound to PRL (grey) in the 2:1 complex (PDB code: 3NPZ), the side view (above) shows the twist between the two FN domains induced by sAB binding and the top view (below) shows a rotation of the FN1 domain relative to the FN2 domain in the sAB-bound structure. d Alignment of the sAB-bound PRL-R (red) with the hGH-bound PRL-R (green) and the PRL-bound PRL-R (yellow) using the FN2. The bottom panel is a top view of the FN1 of all three structures, with the sAB-bound receptor more resembling the hGH-bound conformation. e Inhibition of sAB binding by hGH or PRL using phage ELISA. Both hGH (left panel) and PRL (right panel) inhibit the interaction between phage-displayed sABs and hPRL-R in a concentration dependent manner. f IC50 values of the hGH or PRL inhibition of the interaction between sABs and hPRL-R calculated from e.
Mentions: To gain insight into the structural basis of the interactions between the sABs and hPRL-R, we determined the crystal structure of the receptor in complex with sAB A8. In the structure, the sAB binds to the opposite face of the ECD away from the hormone-binding site (Figure 1a). Interactions between the two molecules are mediated by side chain residues of the CDR loops of the sAB and the hinge region that connects the two tandem fibronectin (FN) domains of hPRL-R (Figure 1b). Alignment of the ECD FN2 domain in the context of the sAB-bound structure with the 2:1 PRLR: PRL structure [8] reveals that the sAB captures a hinge-bending conformation that results in a subtle, but significant rotation of FN1 relative to FN2 (Figure 1c).Figure 1

Bottom Line: This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding.We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA. srizk@nd.edu.

ABSTRACT

Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response.

Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.

Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

Show MeSH
Related in: MedlinePlus