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2

Figure 3 Polypeptide Segments of IR Showing Altered H/D Exchange Profiles upon Ligand Binding and their Possible Burial upon Ligand Binding Show full caption (A) Asp496, Arg498, and Asp499 (within FnIII-1) lie adjacent to Glu453 and Arg454 (within L2 of the opposite monomer). (B) Residues 519–525 of the FnIII-1 CC′ loop lie adjacent to a conserved hydrophobic pocket on L2. (C) Residues 655–658 of the ID lie in proximity to the L1 surface. Residues described are in stick representation, while the molecular surface of amides of residues demonstrating altered H/D exchange profiles is in black and indicated by green arrows.

Our revised IR ectodomain structure offers some insight into IR conformational change upon ligand binding. First, the revised structure exhibits enhanced electrostatic complementarity (with respect to 2DTG/3LOH) across the sparsely packed inter-monomer L2/FnIII-1 interface. L2 residues Arg454 and Glu453 and FnIII-1 residues Asp496, Arg498, and Asp499 appear capable of forming inter-domain electrostatic interactions upon slight relative domain rotation ( Figure 3 A ). In support of this, we note that peptides containing these residues gain protection from hydrogen/deuterium (H/D) exchange upon ligand binding to both IR and IGF-1R (), suggesting that slight reorientation could indeed bring these opposing residues into contact. Second, a previously overlooked feature of 2DTG/3LOH (retained here) is L2 residue Asp464's juxtaposition with its symmetry-related counterpart in an apparent carboxyl-carboxylate pair. Such pairs are often associated with a pH-dependent conformational switch (), and such a role for Asp464 (highly conserved in IR across species) appears consistent with data suggesting that high-affinity insulin binding involves deprotonation of a single IR acid residue (). Known mutations of other residues close to Asp464, namely K460E () and N462S (), are associated with defects in pH-dependent dissociation. Lys460, Asn462, and Asp464 lie in the final turn of L2 and in proximity to the two-fold axis at the apex of the ectodomain, well away from the insulin binding sites (). Third, the revised location of conserved residues 519–529 (within the CC′ loop of FnIII-1, containing the Cys524-Cys524 disulfide) is intriguing. In both IR and IGF-1R, a peptide encompassing L2 residues 344–350 (IR numbering), gains protection from H/D exchange upon ligand binding (): solvent-exposed backbone amides of this peptide map to the border of a conserved hydrophobic pocket on the surface of L2. The N-terminal region (residues 519–529) of the remodeled FnIII-1 CC′ loop now lies in immediate proximity to this pocket within the same monomer, with Phe518 (Tyr503 in IGF-1R) positioned ∼10 Å away ( Figure 3 B). We speculate that, upon hormone binding, conformational change within the receptor head may dock the FnIII-1 CC′ loop into this pocket. Taken together, the above three observations suggest only a slight rearrangement within the (L2-FnIII-1)head module upon ligand binding.