DESIGN OF A MINIMAL HOMEODOMAIN - THE N-TERMINAL ARM MODULATES DNA-BINDING AFFINITY AND STABILIZES HOMEODOMAIN STRUCTURE

This report investigates the sequence specificity requirements for hom eodomain structure and DNA binding activity by the design and synthesi s of a ''minimAl'' homeodomain (for minimalist design and alanine scan ning mutagenesis) which contains the consensus residues and in which a ll nonconsensus residues have bees replaced with alanine. The murine h omeodomain Msx served as the prototype for the minimAl homeodomain, Al a-Msx. We show that Ala-Msx binds to DNA specifically, albeit with low er affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx (NT), which contains a native rather than an alanine-substituted N-ter minal arm, has similar DNA binding affinity as Msx. We show that the n ative N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an int act N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appro priate tertiary structure of Msx, Therefore, the homeodomain ''scaffol d'' consists of consensus residues, which are sufficient for DNA recog nition, and nonconsensus residues in the N-terminal arm, which are req uired for optimal DNA binding affinity and appropriate tertiary struct ure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.