Supplemental Experimental Procedures

Binding studies were performed employing a Biacore 3000 biosensor system, and analysis was carried out using the included BiaEvaluation software. Biotinylated compounds MV1-B and BV6-B were captured on a SA sensor chip, following the manufacturer’s standard protocol. Biotin was captured in a control channel to assess non-specific binding, and used to correct observed binding to MV1-B and BV6-B. SPR analysis was performed in phosphate buffered saline (PBS), 250 mM NaCl, 0.05 % (v/v) Tween 20, 0.01 % (v/v) NaN3, pH 7.4 at 25 °C, with a flow rate of 40 μL/min. Samples were analyzed both with and without 200 μM TCEP. Regeneration of the surface of the SA sensor chip was accomplished by application of 50 mM NaOH and 1 M NaCl. Interaction constants were determined by performing nonlinear fitting of data corrected for bulk refractive index changes. Data were fitted to various models available within the software. Accuracy of the fits was judged from residual plots and statistical parameters. For binding of the single domain (BIR2 or BIR3) constructs of XIAP and c-IAP1 to immobilized MV1-B, a model allowing for conformational rearrangement after