Diverse DTPA-chelated lanthanides with relevance for nuclear medicine bound to an engineered lipocalin reveal conserved ligand geometry

The Anticalin CL31d, an engineered lipocalin protein previously designed to specifically bind rare-earth and related metal ions as chelate complexes with p -NH 2 -Bn-CHX-A″-DTPA (DTPA-NH 2 ), was subjected to structural and binding studies with a series of 11 different M III ·DTPA-NH 2 complexes. These complexes include various lanthanide and main-group metal(III) ions whose radioisotopes are useful in nuclear medicine, with ionic radii ranging from 0.62 to 1.03 Å. Binding activities of the Anticalin for the M III ·DTPA-NH 2 complexes were quantified by fluorescence titration (probing the intrinsic Tyr/Trp emission), revealing K d values of 0.8–2.4 n M for most of the lanthanide ions investigated ( r ion = 0.75–0.96 Å), but showing markedly reduced affinities towards the small and large main-group metal ions Ga 3+ ( K d = 15.3 nM) and Bi 3+ ( K d = 47.8 n M ), respectively. The crystal structures of six representative M III ·DTPA-NH 2 complexes bound to the Anticalin were solved at high resolution (1.5–1.8 Å) using synchrotron X-ray diffraction. Superposition onto the previously described Anticalin CL31 with bound Y 3+ ·DTPA-SCN indicated an essentially invariant conformation both for the binding protein and its metal-chelate ligands, including conserved hydrogen bonds, and a surprisingly uniform ninefold metal coordination via five carboxylate groups, three N atoms and one water molecule. However, there were two exceptions: the small Sc 3+ ion appeared to be coordinated only eightfold with the DTPA-NH 2 chelator but lacking the water ligand, whereas no metal electron density was observed for the Ga 3+ ion, in line with its known noncanonical DTPA complex geometry. Interestingly, in this case a water molecule was detected at the expected position of the central metal ion within the protein-bound DTPA-NH 2 chelator. Our investigation of the influence of chelate geometry on complex stability establishes the Anticalin CL31d as a small and robust universal binding protein for medically relevant M III ·DTPA complexes with surprisingly broad tolerance towards varying ionic radii, thus enabling flexible radionuclide-targeting strategies in nuclear medicine.