Human Cellular Retinol Binding Protein II Forms a Domain-Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering.

Domain-swapping is a mechanism for evolving new protein structure from extant scaffolds, and has been an efficient protein engineering strategy for tailoring functional diversity. However, domain-swapping can only be exploited if it can be controlled, especially in cases where various folds can coexist. Herein, we describe the structure of a domain-swapped trimer of the iLBP family member, hCRBPII, and suggest a mechanism for domain-swapped trimerization. It is further shown that domain-swapped trimerization can be favored by strategic installation of a disulfide bond, demonstrating a strategy for fold control. We further show the domain-swapped trimer to be a useful protein design template by installing a high-affinity metal binding site with the introduction of a single mutation, taking advantage of its 3-fold symmetry. Together, these studies show how one can promote oligomerization, stabilize a specific oligomer, and generate new function with minimal changes to the protein sequence.