Association properties and unfolding of a ßγ-crystallin domain of a Vibrio-specific protein.

The ßγ-crystallin superfamily possesses a large number of versatile members, of which only a few members other than lens ßγ-crystallins have been studied. Understanding the non-crystallin functions as well as origin of crystallin-like properties of such proteins is possible by exploring novel members from diverse sources. We describe a novel ßγ-crystallin domain with S-type (Spherulin 3a type) Greek key motifs in protein vibrillin from a pathogenic bacterium Vibrio cholerae. This domain is a part of a large Vibrio-specific protein prevalent in Vibrio species (found in at least fourteen different strains sequenced so far). The domain contains two canonical N/D-N/D-X-X-S/T-S Ca(2+)-binding motifs, and bind Ca(2+). Unlike spherulin 3a and other microbial homologues studied so far, ßγ-crystallin domain of vibrillin self-associates forming oligomers of various sizes including dimers. The fractionated dimers readily form octamers in concentration-dependent manner, suggesting an association between these two major forms. The domain associates/dissociates forming dimers at the cost of monomeric populations in the presence of Ca(2+). No such effect of Ca(2+) has been observed in oligomeric species. The equilibrium unfolding of both forms follows a similar pattern, with the formation of an unfolding intermediate at sub-molar concentrations of denaturant. These properties exhibited by this ßγ-crystallin domain are not shown by any other domain studied so far, demonstrating the diversity in domain properties.

Evolutionary remodeling of ßγ-crystallins for domain stability at cost of Ca2+ binding.

The topologically similar ßγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in ßγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of ßγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across ßγ-crystallin members critical for their specialized functions.