A plethora of both experimental and computational methods have been proposed in the past 20 years for the identification of hot spots at a protein-protein interface. The experimental determination of a protein-protein complex followed by alanine scanning mutagenesis, though able to determine hot spots with much precision, is expensive and has no guarantee of success while the accuracy of the current computational methods for hot-spot identification remains low. Here, we present a novel

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... -based computational approach that accurately determines hot spots through docking into a set of proteins homologous to only one of the two interacting partners of a compound capable of disrupting the protein-protein interaction (PPI). This approach has been applied to identify the hot spots of human activin receptor type II (ActRII) critical for its binding toward Cripto-I. The subsequent experimental confirmation of the computationally identified hot spots portends a potentially accurate method for hot-spot determination in silico given a compound capable of disrupting the PPI in question. The hot spots of human ActRII first reported here may well become the focal points for the design of small molecule drugs that target the PPI. The determination of their interface may have significant biological implications in that it suggests that Cripto-I plays an important role in both activin and nodal signal pathways.