Fig. 2.
Experimental validation. (A) Screening, selection, and KD measurement by SPR. (Top) The five indicated designs were tested for binding to BCL-xL (immobilized on a biosensor chip). (Bottom) The KD of the design that exhibited the highest binding signal (4H_αBM_1) was determined by performing SPR at the indicated concentrations. Raw data (black lines) were fitted (red lines). (B) Size-exclusion chromatography. 1:1 mixtures of both 4H_αBM_1:BCL-xL and 4H_αBM_1:MCL-1 were eluted as if they were a 1:1 heterodimer. A 1:1:1 mixture of 4H_αBM_1:BCL-xL:MCL-1 was eluted as a heterotrimer with a 1:1:1 binding stoichiometry. The black arrows indicate the elution positions of the standard size markers. (C) Crystal structure of 4H_αBM_1–BCL-xL. (Left) BCL-xL binds both sides of 4H_αBM_1. (Middle) Superposition of 4H_αBM_1–BCL-xL and the design model. The RMSD for the aligned Cα atoms is 1.062 Å. (Right) Magnified views show the preserved hotspot residues in the binding interface: Ile143, Ala144, Leu147, Ile150, Gly151, Asp152, and Phe154. (D) Conformational change. α3 is partially unwound and α2 is elongated in the crystal structure compared to the designed model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)