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Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist

DOI: 10.1073/pnas.1513023113 DOI Help

Authors: Susana Frago (University of Oxford) , Ryan D. Nicholls (University of Bristol) , Madeleine Strickland (University of Bristol) , Jennifer Hughes (University of Oxford) , Christopher Williams (University of Bristol) , Lee Garner (University of Oxford) , Mirvat Surakhy (University of Oxford) , Rory Maclean (University of Oxford) , Dellel Rezgui (University of Oxford) , Stuart N. Prince (University of Oxford) , Oliver J. Zaccheo (University of Oxford) , Daniel Ebner (University of Oxford) , Sabina Sanegre (University of Oxford) , Sheng Yu (University of Oxford) , Francesca M. Buffa (University of Oxford) , Matthew P. Crump (University of Bristol) , Andrew Bassim Hassan (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 113 , PAGES E2766 - E2775

State: Published (Approved)
Published: May 2016
Diamond Proposal Number(s): 8922

Abstract: Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

Journal Keywords: growth factor receptor; protein evolution; insulin-like growth factor 2; binding kinetics; biological therapy

Subject Areas: Biology and Bio-materials, Medicine

Instruments: I04-Macromolecular Crystallography

Added On: 07/12/2016 17:01

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