Bioengineering of human thyrotropin superactive analogs by site-directed "lysine-scanning" mutagenesis - Cooperative effects between peripheral loops

We have previously engineered the first superactive analogs of human thyrot ropin (hTSH) by using a novel design strategy. In this study, we have appli ed homology comparisons focusing on the alpha L3 loop of the common alpha-s ubunit of human glycoprotein hormones. Seven highly variable amino acid res idues were identified, and charge-scanning mutagenesis revealed three previ ously unrecognized modification permissive domains and four gain-of-functio n lysine substitutions. Such gain-of-function mutations were hormone- and r eceptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mut ants, In combinations of the most potent alpha L3 loop analog with two prev iously characterized loop analogs, a higher degree of cooperativity for the alpha L3 loop analog compared with both the alpha L1 loop analog and the h TSH-beta L3 loop analog was observed. We demonstrated that spatially distin ct regions of the common alpha-subunit contribute differentially to the int eraction of hTSH with its receptor and that combinations of two modified lo ops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three sup eractive loops showed cooperativity in receptor binding and activation resu lting in the most potent hTSH superactive analog described to date.