Computational Design of Two Mutant Erythropoietin Receptors that Bind Epo in a Specific Heterodimeric State

Mala Radhakrishnan, Massachusetts Institute of Technology

Erythropoietin (Epo) is a cytokine that induces the maturation of red blood cell precursors, and it is commonly administered to patients with anemia and other conditions. Functionally, Epo binds to two molecules of Erythropoitein receptor (EpoR) at the surface of cells. The EpoR molecules are bound at distinct binding sites on Epo, termed site 1 and site 2. Upon binding Epo, the two EpoR monomers are believed to assume a relative orientation that allows for activation and signal generation. The individual EpoR monomers are identical when unbound from Epo, but because Epo is asymmetric, the two monomers are structurally distinct in the bound-state complex and may therefore play different functional roles in signal generation. We wished to explore the importance of this asymmetry by elucidating each monomer’s individual role in the Epo-EpoR complex. To that end, we computationally designed an experimental system that would allow for separate tracking and modification of EpoR molecules bound at each distinct binding site on Epo. Our computational methods involved a physics-based model and combinatorial algorithms that can predict the structures of mutant complexes as well as the change in the binding free energy of mutation. The designed system consisted of two mutant EpoR molecules, each of which was computationally predicted to bind Epo specifically at exactly one site. Together, these two EpoR molecules were designed to form a specific heterodimeric complex with Epo. Experimental testing of these mutants has validated the computational predictions, and this experimental system is currently being used to understand the precise mechanism by which the receptor monomers begin the signaling process upon binding Epo. The insight gained from this experimental system may be useful in the design of improved Epo-based therapeutics.

Abstract Author(s): Mala L. Radhakrishnan, Xiaohui Lu, Yingxin Lucy Zhang, Alec W. Gross, Harvey F. Lodish, and Bruce Tidor