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 k off 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.growth factor receptor | protein evolution | insulin-like growth factor 2 | binding kinetics | biological therapy T he functional evolution of proteins is largely considered to occur by chance, frequently because of unpredictable and specific events that confer a structure-based change in function sufficient for subsequent selection or "gain of fitness" (1). One such evolutionary biochemical example is the initial acquisition and subsequent gain of affinity between the insulin-like growth factor 2 (IGF2) ligand and a single domain of a nonsignaling mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R) (domain 11). The structural and functional basis of this evolutionary path, which has occurred over 150 million years of mammalian evolution, has been reported previously (2). The questions that we address in the present work are whether the IGF2:domain 11 interaction has reached an optimal state in the context of IGF2 activation of signaling receptors and in the ligand clearance function of M6P/IGF2R, and how far can we extend the binding interaction in terms of structural, biophysical, and functional properties.Functionally, and unlike products of other mammalian imprinted genes, domain 11 is unusual because it specifically evolved to bind to an evolutionary conserved IGF2 ligand with high affinity (3-5). After binding, clearance of extracellular IGF2...
The cation-independent mannose 6-phosphate (M6P)/ Insulin-like growth factor-2 receptor (CI-MPR/ IGF2R) is a ~300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and the internalisation of IGF2. Insulin-like growth factor 2 (IGF2). The extracellular region of the CI-MPR is composed of 15 has fifteen homologous domains including M6P binding domains (D) D3, D5, D9 and D15 and IGF2 binding D11. but how it interacts with extracellular ligands at neutral pH is poorly understood. We have focused structural work on key CI-MPR domains of human CI-MPR and report the first structures of human D7, D8, D9 and D10 within two multi-domain constructs, D9-10 and D7-11. Together These structures provide the first high-resolution description of the high-affinity M6P binding domain D9. Domain 9 stabilises a well-defined hub formed by D7-11 in which whereby two penta-domains intertwine to form a dimeric helical-type coil. Remarkably the D7-11 ligand free structure of this penta-domain closely matches the IGF2 bound state suggesting this may be an intrinsically stable conformation at neutral pH. An Interdomain clusters of histidine and proline residues identified between several pairs of domains impart rigidity and may impart receptor rigidity and play a role in structural transitions of the receptor at low pH.
Insulin-like growth factor 2 (IGF2) plays an important role both in human and mouse embryonic development and in tumour growth. IGF2 causes IGF1R and insulin receptor isoform A, IR(A), dependent activation of proliferation (MAPK) and survival pathways (AKT-PI3K). IGF2 bioavailability is tightly regulated by six IGF binding proteins and by the IGF2/mannose 6-phosphate receptor (IGF2R). IGF2R is a 15-domain extracellular receptor, frequently mutated in cancer, which clears IGF2 via binding to domain 11 and internalising it for lysosomal degradation. Increased supply of IGF2 occurs in hepatocellular, breast, prostate, colorectal and ovarian tumours. Excessive amounts of IGF2 secreted by some tumours, mainly sarcoma, can lead to activation of IR(A) and cause non-islet cell tumour induced hypoglycaemia. Approaches aiming to counteract the activity of IGF2 by targeting IGF1R often fail due to IGF2-mediated activation of IR(A). A ligand trap that exploits the high specificity and affinity of IGF2R for IGF2 has been designed by the fusion of a mutated form of human domain 11 of IGF2R to a C-terminal human IgG1 Fc domain (Fc-dom11). For the ligand trap to function as a clinically useful IGF2 super-antagonist, the binding affinity of domain 11 to IGF2 was further improved. The binding affinity optimisation strategy consisted of random mutagenesis of domain 11 loops involved in IGF2 binding, followed by yeast surface display in P. Pastoris, FACS and SPR screening, as well as structure-directed mutagenesis, complemented with NMR studies. Several mutants showing enhanced IGF2 binding have been identified, the combination of separately identified mutations improving affinity up to 100 fold. In particular, the mutant domain 113-4D shows an increase in affinity through both on- and off-rate modification attributed to the decrease in flexibility of one of the loops involved in IGF2 binding, as suggested by NMR results. The ability of Fc-dom113-4D to function as an IGF2 antagonist has been tested in vitro, using hepatocellular carcinoma cell lines, and in vivo, where it counteracts IGF2-induced hypoglycaemia in mice. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A246. Citation Format: Susana Frago, Madeleine Strickland, Jennifer Hughes, Christopher Williams, Ryan Nicholls, Lee Garner, Dellel Rezgui, Matthew P. Crump, A. Bassim Hassan. Development of an IGF2 super-antagonist by directed evolution of IGF2R. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A246.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.