The Phox homology domain (PX domain) is a phosphoinositide-binding structural domain that is critical in mediating protein and cell membrane association and has been found in more than 100 eukaryotic proteins. The abundance of PX domains in nature offers an opportunity to redesign the protein using EvoDesign, a computational approach to design new sequences based on structure profiles of multiple evolutionarily related proteins. In this study, we report the X-ray crystallographic structure of a designed PX domain from the cytokine-independent survival kinase (CISK), which has been implicated as functioning in parallel with PKB/Akt in cell survival and insulin responses. Detailed data analysis of the designed CISK-PX protein demonstrates positive impacts of knowledge-based secondary structure and solvation predictions and structure-based sequence profiles on the efficiency of the evolutionary-based protein design method. The structure of the designed CISK-PX domain is close to the wild-type (1.54 angstrom in C-alpha RMSD), which was accurately predicted by I-TASSER based fragment assembly simulations (1.32 angstrom in C-alpha RMSD). This study represents the first successfully designed conditional peripheral membrane protein fold and has important implications in the examination and experimental validation of the evolution-based protein design approaches. (C) 2015 Elsevier Inc. All rights reserved.