The N-terminal domain of βb2-crystallin resembles the putative ancestral homodimer

βγ-crystallins from the eye lens are proteins consisting of two similar domains joined by a short linker. All three-dimensional structures of native proteins solved so far reveal similar pseudo-2-fold pairing of the domains reflecting their presumed ancient origin from a single-domain homodimer. However, studies of engineered single domains of members of the βγ-crystallin superfamily have not revealed a prototype ancestral solution homodimer. Here we report the 2.35 Å X-ray structure of the homodimer of the N-terminal domain of rat βB2-crystallin (βB2-N). The two identical domains pair in a symmetrical manner very similar to that observed in native βγ-crystallins, where N and C-terminal domains (which share ∼35% sequence identity) are related by a pseudo-2-fold axis. βB2-N thus resembles the ancestral prototype of the βγ-crystallin superfamily as it self-associates in solution to form a dimer with an essentially identical domain interface as that between the N and C domains in βγ-crystallins, but without the benefit of a covalent linker. The structure provides further evidence for the role of two-domain pairing in stabilising the protomer fold. These results support the view that the βγ-crystallin superfamily has evolved by a series of gene duplication and fusion events from a single-domain ancestor capable of forming homodimers.