The βγ-Crystallin Superfamily Contains a Universal Motif for Binding Calcium

The beta gamma-crystallin superfamily consists Of evolutionarily related proteins with domain topology similar to lens beta- and gamma-crystallins, formed from duplicated Greek key motifs. Ca2+ binding Was Found In a few beta gamma-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well Studied. To increase our understanding of Ca2+ binding in various beta gamma-crystallins, we undertook comprehensive structural and Ca2+-binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca2+ coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca2+ elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca2+-binding abilities are partly or completely compromised. We show that reduction or loss of Ca2+ binding in members of the superfamily, particularly in vertebrates, is due to the selective presence Of unfavorable amino acids (largely Arg) at key Ca2+-ligation positions and that engineering of the canonical Ca2+-binding residues can confer binding activity oil an otherwise inactive domain. Through this work, we demonstrate that beta gamma-crystallins with the N/D-N/D-#-I-S/T-S motif form an extensive set of Ca2+-binding proteins prevalent in all of the three kingdoms of life.