The growing availability of complex structures in the Protein Data Bank has provided key insight into the molecular architecture of protein–protein interfaces. The remarkable diversity observed in protein binding modes is paralleled by a tremendous variation in binding affinities, with interaction half-lives ranging from days to milliseconds. Within the protein interactome, low-affinity binding events have been particularly difficult to visualize by traditional structural methods, which has spurred the development of innovative strategies for reconstituting these short-lived yet biologically essential assemblies. An important takeaway from structural studies of low-affinity systems is that there is no universal solution for stabilizing protein complexes, and approaches such as single-chain fusions, biochemical linkages, and affinity-maturation have each been successful in certain contexts. In this article, we review how advances in molecular engineering have been used to capture weakly associated complexes for structure determination, and we provide perspectives on how the continued application of these methods can shed new light on the “hidden world” of low-affinity interactions.
Impact statement: Low-affinity protein interactions, while biologically essential, have been difficult to visualize by traditional methods in structural biology. In this review, we describe a series of innovative molecular engineering strategies that have been used to stabilize weakly bound protein complexes for structure determination. By highlighting several examples from the literature along with potential advantages and disadvantages of the individual approaches, we hope to provide an introductory resource for structural biologists studying low-affinity systems.
Keywords: Structural biology; X-ray crystallography; cryoelectron microscopy; engineering; nuclear magnetic resonance; protein–protein interactions.