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-NH2-Bn-CHX-A″-DTPA (DTPA-NH2), was subjected to structural and binding studies with a series of 11 different MIII·DTPA-NH2 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 MIII·DTPA-NH2 complexes were quantified by fluorescence titration (probing the intrinsic Tyr/Trp emission), revealing Kd values of 0.8-2.4 nM for most of the lanthanide ions investigated (rion = 0.75-0.96 Å), but showing markedly reduced affinities towards the small and large main-group metal ions Ga3+ (Kd = 15.3 nM) and Bi3+ (Kd = 47.8 nM), respectively. The crystal structures of six representative MIII·DTPA-NH2 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 Y3+·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 Sc3+ ion appeared to be coordinated only eightfold with the DTPA-NH2 chelator but lacking the water ligand, whereas no metal electron density was observed for the Ga3+ 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-NH2 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 MIII·DTPA complexes with surprisingly broad tolerance towards varying ionic radii, thus enabling flexible radionuclide-targeting strategies in nuclear medicine.