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.

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