Delineating the conformational features of intrinsically disordered proteins (IDPs) is an area of work that challenges current experimental and simulation protocols. It is therefore imperative to combine multiple methodologies to arrive at a coherent picture of the heterogeneous IDP ensembles. Here, we present a comprehensive study drawing from structure-based statistical mechanical model, explicit-solvent MD and implicit-solvent REMD simulations, and mutational analysis to characterize, in combination with experimental observables, the functional landscape of the intrinsically disordered DNA-binding domain (DBD) of the Escherichia coli transcription regulator CytR in its free-state. The resulting landscape of CytR DBD populates multiple distinct conformations and shows signs of residual frustration from native interactions that are critical for promoting partial structure in the DNA-binding second helix. We thus find evidence for a unique combination of conformational selection and induced-fit mechanism that enables CytR to bind DNA. Functionally deficient mutations affect the IDP landscape in a nontrivial manner highlighting the importance of using an ensemble-based structural representation of IDPs. In effect, our results reveal a functional role for structural frustration, and provide a novel avenue to characterize IDP landscapes combining simple structural probes and multimodel approaches. © 2013 American Chemical Society.