Phosphorylation-induced conformational changes and functional regulation of the muscle-atrophy-associated ubiquitin ligase Cbl-b

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To elucidate the structural basis for enhancement of Cbl-b’s ubiquitin ligase activity through the phosphorylation, we initially constructed a protein expression system for Cbl-b^<39-465>Y363E mutant that mimics the phosphorylated state of Cbl-b. We succeed in the development of a protein purification protocol in order to generate pure protein in sufficient quantities for X-ray crystallographic studies. We carried out crystal screening of a Cbl-b^<39-465>Y363E purified by this protocol. However, Huang’s group reported the crystal structure of the phosphorylated c-Cbl, a homolog of Cbl-b, in January 2012. Thus, we changed the originally planned research project for the study focused on the interaction between the Cbl-b^<39-341>(hereafter called "Cbl-b TKB") and the phosphorylated IRS-1. We succeeded in the crystal structure determination of the Cbl-b TKB in complex with the short phosphopeptide mimetic of phospholylated IRS-1. It revealed the details of the binding mode of Cbl-b TKB to the phosphopeptide.